Global regulator of secondary metabolite biosynthesis and methods of use

ABSTRACT

The invention provides polypeptides and nucleic acids which identify and encode LaeA, a regulator of fungal secondary metabolite production which exhibits global control over secondary metabolite biosynthetic gene clusters. The invention further provides expression vectors, host cells, methods of increasing the production of secondary metabolites in an organism naturally producing a secondary metabolite or engineered to produce a secondary metabolite, and methods of identifying novel secondary metabolite biosynthesis gene clusters.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/413,073, filed on Sep. 24, 2002, which isincorporated herein by reference in its entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

[0002] This work was supported in part by a grant from the NationalScience Foundation MCB-9874646. The Government of the United States ofAmerica may have certain rights in this invention.

FIELD OF THE INVENTION

[0003] This invention relates generally to the field of secondarymetabolite production in fungi. In particular, this invention isdirected to a gene encoding a regulator of secondary metabolitebiosynthesis and methods of using the same.

BACKGROUND OF THE INVENTION

[0004] Secondary metabolites display a broad range of useful antibioticand immunosuppressant activities as well as less desirable phyto- andmycotoxic activities. (Demain, A., and Fang, A. (2000) The NaturalFunctions of Secondary Metabolites. In Advances in BiochemicalEngineering/Biotechnology, T. Scheper, ed. (Berlin Heidelberg,Springer-Verlag), pp. 1-39). For example, penicillin and derivatives,produced by Aspergillus, Cephalosporium and Penicillium species arewidely used antibiotics (Brakhage, A. A. (1998) Microbiol. Mol. Biol.Rev. 62, 547-585), lovastatin is a potent cholesterol lowering drugproduced by Aspergillus terreus (Kennedy et al. (1999) Science 284,1368-1372) and aflatoxins, produced by several Aspergillus species, arehighly toxic carcinogens contaminating many crops (Hicks et al. (2002)Genetics and Biosynthesis of Aflatoxins and Sterigmatocystin. in TheMycota, Vol. XI, Kempken and Bennett, eds. (Spring-Verlag). Thedistribution of natural products is characteristically restricted tocertain fungal taxa, particularly the Ascomycetes. Perhaps the greatestnumber of known secondary metabolites has been ascribed to theAscomycete genus Emericella (asexual stage=Aspergillus). (reviewed inBrakhage, 1998 and Hicks et al., 2002 respectively).

[0005] Much of the current understanding of fungal secondary metaboliteregulation arises from studies of the genetic model Aspergillusnidulans. This organism produces many natural products includingsterigmatocystin ST (ST; the penultimate precursor to aflatoxin) (Hickset al., 2002) and penicillin (Brakhage, 1998; Penalva et al. (1998)Trends Biotechnol. 16, 483-489) and has been used as a heterologous hostto study the biosynthesis of other natural products including lovastatin(Kennedy et al., 1999). Critical advances in understanding of fungalsecondary metabolism have been largely based on primary studies from A.nidulans and/or secondary studies in other fungi where researchers wereable to exploit the knowledge gained from A. nidulans to their fungus ofchoice (Tag et al. (2000) Mol. Microbiol. 38, 658-665; Borgia et al.(1994) FEMS Microbiol. Lett. 122, 227-231; Shen et al. (1998) Genetics148, 1031-1041). These advances include the discovery of a penicillin(Montenegro et al. (1992) J. Bacteriol. 174, 7063-7067) and STbiosynthetic gene cluster (Brown et al. (1996) Proc. Natl. Acad. Sci.USA 93, 1418-1422) and the establishment of a G-protein/cAMP/proteinkinase A mediated growth pathway in A. nidulans regulating secondarymetabolism production and sporulation (Hicks et al., 1997; Tag et al.,2000; Shimizu et al. (2001) Genetics 157, 591-600).

[0006] Through the use of Aspergillus nidulans, it is now apparent thatstructural genes required for most secondary metabolites are clustered(Keller et al. (1997) Fungal Genetics and Biology 21, 17-29), that theregulation of the clustered genes is largely dependent on pathwayspecific transcription factors (Fernandes et al. (1998) Mo. Microbiol28, 1355-1365; Hohn et al (1999) Fungal Genet. Biol. 26, 224-235; Tsujiet al. (2000) Mol. Microbiol. 38, 940-954) and that G protein regulationof fungal secondary metabolism is likely to be a conserved phenomena(Tag et al., 2000) presumably transmitted through the pathway specifictranscription factor.

[0007] The biosynthetic genes necessary for sterigmatocystin (ST)production in A. nidulans are clustered on a ca. 60-kb region onchromosome IV (Brown et al., 1996). The expression of these clustergenes (called stc genes) is regulated by the sixth gene in the cluster,aflR. aflR encodes a zinc binuclear cluster DNA binding protein whichbinds to AflR sites in stc promoters (Fernandes et al., 1998). ST is thepenultimate precursor of aflatoxin (AF), which is produced by therelated species A. flavus and A. parasiticus. AFlR was first identifiedin A. flavus (Payne et al. (1993) Appl. Environ Microbiol. 59, 156-162)and subsequently in A. parasiticus (Chang et al. (1993) Appl. Environ.Microbiol. 59, 3273-3279). AflR regulates the expression of the AFcluster genes in both A. flavus and A. parasiticus in a manner similarto the stc genes. aflR is not constitutively expressed in these threespecies and is regulated through a complex interaction with Gprotein/cAMP/protein kinase A signal transduction pathway also involvedin asexual spore development (Hicks et al., 1997; Shimizu and Keller,2001).

[0008] The discovery of G protein/cAMP/protein kinase A regulation of STand other fungal secondary metabolites (Shimizu and Keller; 2001; Hickset al., 2002; Tag et al., 2000) has been decidedly helpful inestablishing a concept of global regulation of secondary metabolism.However, currently available signal transduction mutants havepleiotrophic effects on the fingi, the most notable effect being thegross impact on spore production and vegetative hyphal growth (Hicks etal., 1997; Tag et al., 2000; Shimizu and Keller, 2001; Adams et al.(1998) Curr. Opin. Microbiol. 1, 674-677). Thus, currently availablesignal transduction mutants are so impaired as to fungal developmentthat further elucidation of genes specific for regulation of secondarymetabolite gene clusters is difficult.

[0009] Studies of bacteria have only recently identified unique proteinswhose primary function appears to be directed to regulation of multiplegroups of secondary metabolism genes. These include AfsR, atranscriptional factor with ATPase activity regulating the production ofactinorhodin, undecylprodigiosin and calcium-dependent antibiotic inStreptomyces coelicolor (Lee et al. (2002) Mol. Microbiol. 43,1413-1430) and RsmA, a post transcriptional regulator of secondarymetabolites and virulence factors in Pseudomonas aeruginosa (Pessi etal. (2001) J. Bacteriol. 183, 6676-6683). Similar proteins have not yetbeen identified in fungi but the existence of such in bacteria suggeststhe exciting possibility of global regulators of secondary metabolism inthe Fungal Kingdom.

[0010] Although various similarities have been observed betweensecondary metabolite gene clusters in terms of cluster-specificregulatory elements, identification of regulatory elements providingglobal regulation of secondary metabolite gene clusters with littleeffect on sporulation and vegetative growth have not been reported. Suchregulatory elements are extremely desirable because they would possessbroad specificity for the activation and/or repression of entirefamilies of secondary metabolite gene clusters while providing strainscapable of otherwise normal or near-normal development and growth.Furthermore, identification of such regulatory elements would enable theincreased production of secondary metabolites by providing improvedstrains of engineered organisms and also contribute to the broaderunderstanding of molecular mechanisms by which secondary metabolites areproduced.

SUMMARY OF THE INVENTION

[0011] The inventors describe and claim herein an archetypal globalregulator of secondary metabolism in fungi, termed LaeA. Deletion of thegene encoding LaeA blocks sterigmatocystin (polyketide carcinogen),penicillin (non-ribosomal peptide antibiotic), lovastatin (polyketideantihypercholesterolemic agent) and mycelial pigment biosynthesis in A.nidulans, and gliotoxin (non-ribosomal peptide immunotoxin) and mycelialpigment biosynthesis in A. fumigatus. In contrast, over expression oflaeA triggers increased penicillin production of 400-900% in A. nidulansand lovastatin product formation of 500-700% in A. terreus,respectively.

[0012] Thusly, the present invention provides an isolated polypeptidecomprising an amino acid sequence selected from the group consisting of:(a) an amino acid sequence which is at least 85% identical to the LaeAamino acid sequence set forth in SEQ ID NO:3; (b) an amino acid sequenceencoded by a nucleic acid comprising the laeA nucleotide sequence setforth in SEQ ID NO:2; and (c) an amino acid sequence encoded by anucleic acid which specifically hybridizes under stringent conditions toeither strand of a denatured, double-stranded nucleic acid comprisingthe laeA nucleotide sequence set forth in SEQ ID NO:2.

[0013] In one preferred embodiment, the isolated polypeptide accordingto the invention possesses secondary metabolite gene cluster regulatingactivity. In more preferred embodiments, the polypeptide possessesregulating activity for the lovastatin or penicillin biosynthesis genecluster.

[0014] In certain embodiments, the polypeptide has proteinmethyltransferase activity. In yet other embodiments, the polypeptidecomprises an amino acid sequence at least 95% identical to the completeLaeA amino acid sequence set forth in SEQ ID NO:3, or a fragmentthereof. In a most preferred embodiment, the polypeptide is the LaeAamino acid sequence set forth in SEQ ID NO:3.

[0015] In yet another aspect, the present invention provides an isolatednucleic acid comprising a nucleotide sequence selected from the groupconsisting of: (a) an laeA nucleotide sequence set forth in SEQ ID NO:2;(b) a nucleotide sequence encoding the LaeA amino acid sequence setforth in SEQ ID NO:3; and (c) a nucleotide sequence which specificallyhybridizes under stringent conditions to either strand of a denatured,double-stranded laeA nucleic acid having a nucleotide sequence set forthin SEQ ID NO:2.

[0016] The invention further provides expression vectors including anisolated nucleic acid as described and claimed herein which is inoperative association with one or more regulatory elements. As well,transformed host cells or organisms comprising an isolated nucleic acidas described and claimed herein are further contemplated by theinvention. In preferred embodiments, transformed host cells or organismsproduce secondary metabolites in increased amounts relative tountransformed cells or organisms. The increased secondary metaboliteproduction is preferably at least two fold greater than that ofuntransformed cells or organisms.

[0017] Also provided by the invention are methods of preparing anisolated polypeptide comprising LaeA or fragments thereof. Such methodsinclude the step of culturing a transformed host cell or organism asdescribed and claimed herein under conditions conducive to expression ofthe polypeptide, and recovering the expressed polypeptide from the cellor organism in isolated form.

[0018] The invention is also directed to methods of detecting a nucleicacid encoding an LaeA amino acid sequence set forth in SEQ ID NO:3 in abiological sample comprising the steps of: (a) hybridizing a complementof a nucleotide sequence which encodes an LaeA amino acid sequence asset forth in SEQ ID NO:3 to a nucleic acid material of a biologicalsample thereby forming a hybridization complex; and (b) detecting thehybridization complex wherein the presence of the complex correlateswith the presence of a nucleic acid encoding an LaeA amino acid sequenceset forth in SEQ ID NO:3.

[0019] The present invention further encompasses methods of increasingthe amount of a secondary metabolite produced in a cell or organism.Such methods include steps of: (a) obtaining a cell or an organismcapable of biosynthesizing a secondary metabolite; (b) transforming thecell or organism with an nucleic acid encoding an LaeA polypeptidecapable of regulating biosynthesis of the secondary metabolite; and (c)culturing the transformed cell or organism so that an increase inproduction of the secondary metabolite occurs in the transformed cell ororganism as compared to a non-transformed cell or organism.

[0020] Preferably, methods of increasing the amount of a secondarymetabolite as described and claimed herein are practiced in anAspergillus species. Even more preferably, the Aspergillus species is A.nidulans or A. terreus. As well, preferred secondary metabolitesincreased by the methods are lovastatin or penicillin.

[0021] The invention also provides methods of decreasing the productionof a secondary metabolite in a transformed cell or organism. Suchmethods include the steps of: (a) obtaining a transformed cell ororganism capable of biosynthesizing a secondary metabolite, thetransformed cell or organism having a defective laeA gene wherein thedefective laeA gene is no longer biologically active and expression ofsecondary metabolite gene clusters is reduced; and (b) culturing thetransformed cell or organism so that a decrease in production of thesecondary metabolite occurs in the transformed cell or organism ascompared to a non-transformed cell or organism.

[0022] In yet another embodiment, the present invention encompassesmethods of producing an isolated secondary metabolite. These methodsinclude steps of: (a) obtaining a cell or an organism capable ofbiosynthesizing a secondary metabolite; (b) transforming the cell ororganism with a nucleic acid encoding an LaeA polypeptide capable ofregulating biosynthesis of the secondary metabolite; (c) culturing thetransformed cell or organism under conditions conducive to increasingproduction of the secondary metabolite in the transformed cell ororganism as compared to a non-transformed cell or organism; and (d)recovering the secondary metabolite from the transformed cell ororganism in an isolated form.

[0023] Finally, the invention provides methods for identifying yetundiscovered secondary metabolite biosynthesis gene clusters in avariety of fingi based on the nucleic acids and transformed cellsdisclosed herein. Such methods are preferably carried out in amicroarray format.

[0024] Other objects, features and advantages of the present inventionwill become apparent after review of the specification, claims anddrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025]FIG. 1. a, Schematic of laeA gene. Although LaeA contains theexact SAM motif found in histone methyltransferases (HMTases) andarginine methyltransferases (PRMTs), it lacks other conserved domains(e.g. SET domain, a double E loop) typically found in these proteins. Inaddition, likely HMTase and PRMT candidates are found in the Aspergillusdatabase (1e⁻⁴²; 1e⁻⁹⁴). Therefore LaeA appears to be a unique proteinmethyltransferase. b, Amino acid comparison of A. nidulans, A.fumigatus, Neurospora crassa, Magnaporthe grisea, Coccidioides immitisand Fusarium sporotrichioides LaeA proteins showing conserved proteinmethyltransferase s-adenosylmethionine binding site in red. c, Loss ofprotein methylation in soluble nuclear extract from the ΔlaeA strain.

[0026]FIG. 2. a, Asexual sporulation (top) and mycelial pigmentation(bottom) patterns of A. nidulans WT (RDIT2.3) and ΔlaeA (RJW46.4)strains after 5 days cultivation on glucose minimal medium (GMM). A.fumigatus ΔlaeA presented a similar loss of mycelial pigmentation (alsosee FIG. 5). b, Thin layer chromatography analysis of organic solventextracts of RDIT2.3 and RJW46.4 after 5 days cultivation on solid GMM.c, Thin layer chromatography analysis of organic solvent extracts of A.fumigatus AF293 (WT) and TJW54.2 (ΔlaeA) grown in liquid shaking GMM for3 days. Experiment was triplicated. ST=sterigmatocystin andStd=gliotoxin standard.

[0027]FIG. 3. laeA regulation of secondary metabolism. a, aflR, stcU andipnA gene expression in A. nidulans WT (RDIT2.3) and ΔlaeA (RJW46.4)grown in liquid shaking GMM for 12h, 24h, 48h and 72h at 37° C. Ethidiumbromide stained rRNA is indicated for loading. b, laeA, lovE and lovCgene expression in A. nidulans WT;lov+ (RJW51) and ΔlaeA;lov+ (RJW53)strains grown in liquid shaking GMM for 12h, 24h, 48h and 72h at 37° C.Ethidium bromide stained rRNA is indicated for loading. MONJ wasextracted from WT; lov+ (RJW51) and ΔlaeA; lov+ (RJW53) A. nidulansstrains grown in liquid shaking GMM for 3 days. Experiment wastriplicated. c, laeA, ipnA, stcU, lovE and lovC gene expression, and STand MONJ extraction in A. nidulans WT (RDIT2.3) and OE::laeA (RJW47.3),and WT:lov+ (RJW51) and OE::laeA;lov+ (RJW52) grown in liquid shakingGMM for 14h at 37° C., and then transferred to liquid shaking threonineminimal media for induction of laeA expression. Time points were 0h, 6h,12h and 24h after transfer. ST and MONJ extracts from A. nidulans WT(RDIT2.3) and OE::laeA (RJW47.3), and WT; lov+ (RJW51) and OE::laeA;lov+ (RJW52) grown in liquid shaking GMM for 14h at 37° C., and thentransferred to liquid shaking threonine minimal media for 24h afterswitch ST=ST standard. MONJ standard was extracted from A. nidulansstrain WMH1739. Experiment was triplicated. d, Penicillin bioassay. Wildtype (FGSC26), ΔlaeA (RJW40.4) and OE::laeA (RJW44.2) were grown inliquid shaking GMM for 14h at 37° C., and then transferred to lactoseminimal medium amended with 30 mM cyclopentanone for induction of laeAfor 24h at 37° C. e, Lovastatin production in A. terreus in laeA overexpression strains. Wild type (ATCC20542, lane 1), TJW58.9 (hygBresistance gene containing transformant as a control, lane 2) andOE::laeA strains containing hygB (TJW58.2, TJW58.4, TJW58.7, TJW58.8 andTJW58.14 are lanes 3, 4, 5, 6 and 7, respectively) were grown in liquidshaking GMM for 18h at 32° C., and then transferred to lactose minimalmedium with 30 mM cyclopentanone for induction of laeA for 36h at 32° C.Std=lovastatin standard. Experiment was duplicated. f, Regulation oflaeA. Effects of over expression of aflR, pkaA and ras^(G17V) on laeAexpression. WT (RKIS1), OE::aflR (TJH34.10), OE::pkaA (TKIS20.1) andOE::ras^(G17V) (RKIS28.5) were grown in liquid shaking GMM for 14h at37° C., and then transferred to threonine minimal medium. Time pointswere 0h, 6h, 12h or 24h after transfer.

[0028]FIG. 4. Schematic of LaeA's putative role as a global regulator ofsecondary metabolisms.

[0029]FIG. 5. Aspergillus nidulans LaeA protein localizes to thenucleus. The green fluorescent protein (GFP) was fused to LaeA and asdescribed herein. Nuclei were stained with the DNA-specific dye4,6-diamindino-2-phenylindole (DAPI).

[0030]FIG. 6. Aspergillus fumigatus transformants. Left is the top viewof the petri dish and right the bottom view. Arrows indicate strainswith a disrupted laeA gene.

[0031]FIG. 7. Transcript analysis of the ST gene cluster and genesimmediately upstream and downstream of the gene cluster. A. nidulans WT(RDIT2.3) and ΔlaeA (RJW46.4) strains were grown in liquid shaking GMMfor 12h, 24h, 48h and 72h at 37° C. stcA and stcU are two characterizedST biosynthetic genes at either end of the ST gene cluster⁴⁸. Ethidiumbromide stained rRNA is indicated for loading.

[0032]FIG. 8. laeA expression is not affected in ΔflbA, ΔsfaD and Δ aflRstrains. laeA, aflR and stcU gene expression in A. nidulans WT (TPK1.1,RKIS1), ΔflbA (TBN39.5), ΔsfaD (TSRB1.38), and Δ aflR (RMFV2) strainsgrown in liquid shaking GMM for 12h, 24h, 48h and 72h at 37° C. Ethidiumbromide stained rRNA is indicated for loading.

[0033]FIG. 9. laeA expression is not affected in fadA mutants. laeA,aflR and stcU gene expression in A. nidulans WT(FGSC26), ΔfadAG42R(H1FAD4) and WT(RKIS1), ΔfadA (RKIS 11.1) strains grown in liquidshaking GMM for 12h, 24h, 48h and 72h at 37° C. Ethidium bromide stainedrRNA is indicated for loading.

DETAILED DESCRIPTION OF THE INVENTION

[0034] I. In General

[0035] Before the present polypeptides, nucleic acids, and methods aredescribed, it is understood that this invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

[0036] It must be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference to“a cell” includes a plurality of such cells, reference to the “vector”is a reference to one or more vectors and equivalents thereof known tothose skilled in the art, and so forth.

[0037] Unless defined otherwise, all technical and scientific terms usedherein have the same meanings as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods, devices, and materials are now described. All publicationsmentioned herein are incorporated herein by reference for the purpose ofdescribing and disclosing the polypeptides, polynucleotides, cell lines,vectors, and methodologies which are reported in the publications whichmight be used in connection with the invention. Nothing herein is to beconstrued as an admission that the invention is not entitled to antedatesuch disclosure by virtue of prior invention.

[0038] The practice of the present invention will employ, unlessotherwise indicated, conventional techniques of molecular biology,microbiology, recombinant DNA, and immunology, which are within theskill of the art. Such techniques are explained fully in the literature.See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. bySambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press:1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985);Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S.Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J.Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J.Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R.Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B.Perbal, A Practical Guide To Molecular Cloning (1984); the treatise,Methods In Enzymology (Academic Press, Inc., N.Y.); Gene TransferVectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987,Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155(Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology(Mayer and Walker, eds., Academic Press, London, 1987); Handbook OfExperimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell,eds., 1986); Cell Culture and Somatic Cell Genetics of Plants, Vol. 1(I. K. Vasil, ed. 1984); R. V. Stanier, J. L. Ingraham, M. L. Wheelis,and P. R. Painter, The Microbial World, (1986) 5th Ed. Prentice-Hall.

[0039] II. Definitions

[0040] “LaeA”, as used herein, refers to the amino acid sequences of theLaeA protein obtained from Aspergillus nidulans. In addition, LaeA shallalso refer to the amino acid sequences of LaeA obtained from any species(i.e., orthologs), particularly fungi (e.g. other strains and/or speciesof Aspergillus, and the like), from any source whether natural,synthetic, semi-synthetic, or recombinant. The term encompasses proteinsencoded by nucleotide sequences representing allelic variants as well asthose containing single nucleotide polymorphisms (SNPs).

[0041] “laeA”, as used herein, refers to the nucleotide sequences of thelaeA gene obtained from Aspergillus nidulans. In addition, laeA shallalso refer to the nucleotide sequences of the laeA gene obtained fromany species, particularly fungi (e.g. other strains and/or species ofAspergillus, and the like), from any source whether natural, synthetic,semi-synthetic, or recombinant. The term encompasses allelic variantsand single nucleotide polymorphisms (SNPs).

[0042] “SEQ ID NO:1” refers to a nucleotide sequence from genomic DNAisolated from A. nidulans which is 3100 nucleotides in length andencompasses coding sequence for the LaeA protein as well as upstream anddownstream genomic DNA sequences. SEQ ID NO:1 is set forth in itsentirety in the Sequence Listing. The LaeA coding sequence extends froma start codon at nt 959 to a stop codon at nt 2213 with a single introninterrupting the coding sequence from nt 1195-1323.

[0043] “SEQ ID NO:2”, set forth in its entirety in the Sequence Listing,refers to cDNA isolated from A. nidulans which is 1125 nucleotides inlength and encompasses the nucleotide sequence encoding the LaeAprotein.

[0044] “SEQ ID NO:3” refers to an amino acid sequence encoding the LaeAprotein of 374 amino acids in length as isolated from A. nidulans andset forth in the Sequence Listing.

[0045] An “allele” or “allelic sequence”, as used herein, is analternative form of the gene encoding LaeA. Alleles may result from atleast one mutation in the nucleic acid sequence and may result inaltered mRNAs or polypeptides whose structure or function may or may notbe altered. Any given natural or recombinant gene may have none, one, ormany allelic forms. Common mutational changes which give rise to allelesare generally ascribed to natural deletions, additions, or substitutionsof nucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

[0046] “Altered” nucleic acid sequences encoding LaeA, as used herein,include those with deletions, insertions, or substitutions of differentnucleotides resulting in a polynucleotide that encodes the same or afunctionally equivalent protein to LaeA. Included within this definitionare polymorphisms which may or may not be readily detectable using aparticular oligonucleotide probe of the polynucleotide encoding LaeA,and improper or unexpected hybridization to alleles, with a locus otherthan the normal chromosomal locus for the polynucleotide sequenceencoding LaeA. The encoded protein may also be “altered” and containdeletions, insertions, or substitutions of amino acid residues whichproduce a silent change and result in a functionally equivalent LaeA.Deliberate amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological or immunological activity of LaeA is retained. Forexample, negatively charged amino acids may include aspartic acid andglutamic acid; positively charged amino acids may include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values may include leucine, isoleucine, andvaline, glycine and alanine, asparagine and glutamine, serine andthreonine, and phenylalanine and tyrosine.

[0047] “Amino acid sequence”, as used herein, refers to an oligopeptide,peptide, polypeptide, or protein sequence, and fragment thereof. Where“amino acid sequence” is recited herein to refer to a particular aminoacid sequence (e.g., the amino acid sequence set forth in SEQ ID NO:3),“amino acid sequence”, and like terms, are not meant to limit the aminoacid sequence to the complete amino acid sequence referenced but shallbe understood to include fragments of the complete amino acid sequence.The term shall further encompass synthetic molecules as well as thoseoccurring naturally. The term “portion” or “fragment”, as used herein,with regard to an amino acid sequence (as in “a fragment of SEQ IDNO:3”), specifically refers to segments of that amino acid sequencewhich are not naturally occurring as fragments and would not be found inthe natural state. The segments may range in size from five amino acidresidues to the entire amino acid sequence minus one amino acid. Thus, apolypeptide “comprising at least a portion of the amino acid sequence ofSEQ ID NO:3” or “including an amino acid sequence as set forth in SEQ IDNO:3 or fragments thereof” encompasses the full-length LaeA amino acidsequences and segments thereof.

[0048] “Amplification”, as used herein, refers to the production ofadditional copies of a nucleic acid sequence and is generally carriedout using polymerase chain reaction (PCR) technologies well known in theart (Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, aLaboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.).

[0049] The term “antisense”, as used herein, refers to any compositioncontaining nucleotide sequences which are complementary to a specificDNA or RNA sequence. The term “antisense strand” is used in reference toa nucleic acid strand that is complementary to the “sense” strand.Antisense molecules include peptide nucleic acids and may be produced byany method including synthesis or transcription. Once introduced into acell, the complementary nucleotides combine with natural sequencesproduced by the cell to form duplexes and block either transcription ortranslation. The designation “negative” is sometimes used in referenceto the antisense strand, and “positive” is sometimes used in referenceto the sense strand.

[0050] The term “biologically active”, as used herein, refers to aprotein, polypeptide, amino acid sequence, or nucleotide sequenceencoding a product having structural, regulatory, or biochemicalfunctions of a naturally occurring molecule. Preferably, a biologicallyactive fragment of LaeA will have the secondary metabolite gene clusterregulatory capabilities of a naturally occurring LaeA molecule disclosedherein.

[0051] The terms “complementary” or “complementarity”, as used herein,refer to the natural binding of polynucleotides under permissive saltand temperature conditions by base-pairing. For example, the sequence“A-G-T” binds to the complementary sequence “T-C-A”. Complementarybetween two single-stranded molecules may be “partial”, in which onlysome of the nucleic acids bind, or it may be complete when totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions,which depend upon binding between nucleic acids strands and in thedesign and use of PNA molecules.

[0052] A “composition comprising a given polynucleotide sequence”, asused herein, refers broadly to any composition containing the givenpolynucleotide sequence. Compositions comprising polynucleotidesequences encoding LaeA (SEQ ID NO:1 or 2) or fragments thereof, may beemployed as hybridization probes. The probes may be stored infreeze-dried form and may be associated with a stabilizing agent such asa carbohydrate. In hybridizations, the probe may be deployed in anaqueous solution containing salts (e.g., NaCl), detergents (e.g., SDS)and other components (e.g., Denhardt's solution, dry milk, salmon spermDNA, etc.).

[0053] The term “correlates with expression of a polynucleotide”, asused herein, indicates that the detection of the presence of ribonucleicacid that is similar to SEQ ID NO:2 by northern analysis or equivalentanalysis is indicative of the presence of mRNA encoding LaeA in a sampleand thereby correlates with expression of the transcript from thepolynucleotide encoding the protein.

[0054] A “deletion”, as used herein, refers to a change in the aminoacid or nucleotide sequence and results in the absence of one or moreamino acid residues or nucleotides.

[0055] The term “derivative”, as used herein, refers to the chemicalmodification of a nucleic acid encoding or complementary to LaeA or theencoded LaeA protein itsef. Such modifications include, for example,replacement of hydrogen by an alkyl, acyl, or amino group. A nucleicacid derivative encodes a polypeptide which retains the biological orimmunological function of the natural molecule. A derivative polypeptideis one which is modified by glycosylation, or any similar process whichretains the biological function of the polypeptide from which it wasderived.

[0056] The term “homology”, as used herein, refers to sequencesimilarity between two peptides or between two nucleic acid molecules.Homology may be determined by comparing a postion in each sequence whichmay be aligned for purposes of comparison. When a position in thecompared sequence is occupied by the same base or amino acid, then themolecules are homologous at that position. A degree of homology betweensequences is a function of the number of matching or homologouspositions shared by the sequences. A partially complementary sequencethat at least partially inhibits an identical sequence from hybridizingto a target nucleic acid is referred to using the functional term“substantially homologous.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or hybridization probe will competefor and inhibit the binding of a completely homologous sequence to thetarget sequence under conditions of low stringency. This is not to saythat conditions of low stringency are such that non-specific binding ispermitted; low stringency conditions require that the binding of twosequences to one another be a specific (i.e., selective) interaction.The absence of non-specific binding may be tested by the use of a secondtarget sequence which lacks even a partial degree of complementary(e.g., less than about 30% identity). In the absence of non-specificbinding, the probe will not hybridize to the second non-complementarytarget sequence. In the art, “identity” means the degree of sequencerelatedness between polypeptide or polynucleotide sequences, as the casemay be, as determined by the match between strings of such sequences.“Identity” and “homology” can be readily calculated by known methods,including but not limited to those described in (Computational MolecularBiology, Lesk, A. M., ed., Oxford University Press, New York, 1988;Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,Academic Press, New York, 1993; Computer Analysis of Sequence Data, PartI, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,1994; Sequence Analysis in Molecular Biology, von Heinje, G., AcademicPress, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux,J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman,D., SIAM J. Applied Math., 48: 1073 (1988). Preferred methods todetermine identity are designed to give the largest match between thesequences tested. Methods to determine identity and homology arecodified in publicly available computer programs. Preferred computerprogram methods to determine identity and homology between two sequencesinclude, but are not limited to, the GCG program package (Devereux, J.,et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, andFASTA (Atschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990). TheBLAST X program is publicly available from NCBI and other sources (BLASTManual, Altschul, S., et al, NCBI NLM NIH Bethesda, Md. 20894; Altschul,S., et al., J. Mol. Biol. 215: 403-410 (1990). The well known SmithWaterman algorithm may also be used to determine identity.

[0057] The term “hybridization”, as used herein, refers to any processby which a strand of nucleic acid binds with a complementary strandthrough base pairing.

[0058] The term “hybridization complex”, as used herein, refers to acomplex formed between two nucleic acid sequences by virtue of theformation of hydrogen bonds between complementary G and C bases andbetween complementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., Co t or Ro tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,paper, membranes, filters, chips, pins or glass slides, or any otherappropriate substrate to which cells or their nucleic acids have beenfixed).

[0059] An “insertion” or “addition”, as used herein, refers to a changein an amino acid or nucleotide sequence resulting in the addition of oneor more amino acid residues or nucleotides, respectively, as compared tothe naturally occurring molecule.

[0060] “Isolated” or “purified” or “isolated and purified” means altered“by the hand of man” from its natural state, i.e., if it occurs innature, it has been changed or removed from its original environment, orboth. For example, a polynucleotide or a polypeptide naturally presentin a living organism is not “isolated,” but the same polynucleotide orpolypeptide separated from the coexisting materials of its natural stateis “isolated”, as the term is employed herein. Moreover, apolynucleotide or polypeptide that is introduced into an organism bytransformation, genetic manipulation or by any other recombinant methodis “isolated” even if it is still present in said organism, whichorganism may be living or non-living. As so defined, “isolated nucleicacid” or “isolated polynucleotide” includes nucleic acids integratedinto a host cell chromosome at a heterologous site, recombinant fusionsof a native fragment to a heterologous sequence, recombinant vectorspresent as episomes or as integrated into a host cell chromosome. Asused herein, the term “substantially purified”, refers to nucleic oramino acid sequences that are removed from their natural environment,isolated or separated, and are at least 60% free, preferably 75% free,and most preferably 90% free from other components with which they arenaturally associated. As used herein, an isolated nucleic acid “encodes”a reference polypeptide when at least a portion of the nucleic acid, orits complement, can be directly translated to provide the amino acidsequence of the reference polypeptide, or when the isolated nucleic acidcan be used, alone or as part of an expression vector, to express thereference polypeptide in vitro, in a prokaryotic host cell, or in aeukaryotic host cell.

[0061] As used herein, the term “exon” refers to a nucleic acid sequencefound in genomic DNA that is bioinformatically predicted and/orexperimentally confirmed to contribute contiguous sequence to a maturemRNA transcript.

[0062] As used herein, the phrase “open reading frame” and theequivalent acronym “ORF” refer to that portion of a transcript-derivednucleic acid that can be translated in its entirety into a sequence ofcontiguous amino acids. As so defined, an ORF has length, measured innucleotides, exactly divisible by 3. As so defined, an ORF need notencode the entirety of a natural protein.

[0063] The term “microarray” refers to an ordered arrangement ofhybridizable array elements. The array elements are arranged so thatthere are preferably at least one or more different array elements, morepreferably at least 100 array elements, and most preferably at least1,000 array elements, on a 1 cm² substrate surface. The maximum numberof array elements is unlimited, but is at least 100,000 array elements.Furthermore, the hybridization signal from each of the array elements isindividually distinguishable. In a preferred embodiment, the arrayelements comprise polynucleotide representative of fungal-derivedpolynucleotide sequences.

[0064] The term “modulate”, as used herein, refers to a change in theactivity of LaeA. For example, modulation may cause an increase or adecrease in protein activity, binding characteristics, or any otherbiological, functional or immunological properties of LaeA.

[0065] “Nucleic acid sequence” or “nucleotide sequence” orpolynucleotide sequence”, as used herein, refers to an oligonucleotide,nucleotide, or polynucleotide, and fragments thereof, and to DNA or RNAof genomic or synthetic origin which may be single- or double-stranded,and represent the sense or antisense strand. Where “nucleic acidsequence” or “nucleotide sequence” or polynucleotide sequence” isrecited herein to refer to a particular nucleotide sequence (e.g., thenucleotide sequence set forth in SEQ ID NO:2), “nucleotide sequence”,and like terms, are not meant to limit the nucleotide sequence to thecomplete nucleotide sequence referenced but shall be understood toinclude fragments of the complete nucleotide sequence. In this context,the term “fragment” may be used to specifically refer to those nucleicacid sequences which are not naturally occurring as fragments and wouldnot be found in the natural state. Generally, such fragments are equalto or greater than 15 nucleotides in length, and most preferablyincludes fragments that are at least 60 nucleotides in length. Suchfragments find utility as, for example, probes useful in the detectionof nucleotide sequences encoding LaeA.

[0066] The term “sample”, as used herein, is used in its broadest sense.A biological sample suspected of containing nucleic acid encoding LaeA,or fragments thereof, or LaeA itself may comprise a bodily fluid,extract from a cell, chromosome, organelle, or membrane isolated from acell, a cell, genomic DNA, RNA, or cDNA (in solution or bound to a solidsupport, a tissue, a tissue print, and the like).

[0067] A “substitution”, as used herein, refers to the replacement ofone or more amino acids or nucleotides by different amino acids ornucleotides, respectively. The term “conservative substitution” is usedin reference to proteins or peptides to reflect amino acid substitutionsthat do not substantially alter the activity (specificity or bindingaffinity) of the molecule. Typically conservative amino acidsubstitutions involve substitution one amino acid for another amino acidwith similar chemical properties (e.g. charge or hydrophobicity). Thefollowing six groups each contain amino acids that are typicalconservative substitutions for one another: 1) Alanine (A), Serine (S),Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine(N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I),Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F),Tyrosine (Y), Tryptophan (W).

[0068] “Transformation”, as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the type of host cell beingtransformed and may include, but is not limited to, viral infection,electroporation, heat shock, lipofection, and particle bombardment. Such“transformed” cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

[0069] A “variant” of LaeA, as used herein, refers to an amino acidsequence that is altered by one or more amino acids. The variant mayhave “conservative” changes, wherein a substituted amino acid hassimilar structural or chemical properties, e.g., replacement of leucinewith isoleucine. More rarely, a variant may have “nonconservative”changes, e.g., replacement of a glycine with a tryptophan. Analogousminor variations may also include amino acid deletions or insertions, orboth. Guidance in determining which amino acid residues may besubstituted, inserted, or deleted without abolishing biological orimmunological activity may be found using computer programs well knownin the art, for example, DNASTAR software.

[0070] The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The term also includes variations on the traditional peptidelinkage joining the amino acids making up the polypeptide. Where theterms are recited herein to refer to a polypeptide, peptide or proteinof a naturally occurring protein molecule, the terms are not meant tolimit the polypeptide, peptide or protein to the complete, native aminoacid sequence associated with the recited protein molecule but shall beunderstood to include fragments of the complete polypeptide. The term“portion” or “fragment”, as used herein, with regard to a protein orpolypeptide (as in “a fragment of the LaeA polypeptide”) refers tosegments of that polypeptide which are not naturally occurring asfragments in nature. The segments may range in size from five amino acidresidues to the entire amino acid sequence minus one amino acid. Thus, apolypeptide “as set forth in SEQ ID NO:3 or a fragment thereof”encompasses the full-length amino acid sequence set forth in SEQ ID NO:3as well as segments thereof. Fragments of LaeA preferably arebiologically active as defined herein.

[0071] The terms “nucleic acid” or “oligonucleotide” or “polynucleotide”or grammatical equivalents herein refer to at least two nucleotidescovalently linked together. A nucleic acid of the present invention ispreferably single-stranded or double stranded and will generally containphosphodiester bonds, although in some cases, as outlined below, nucleicacid analogs are included that may have alternate backbones, comprising,for example, phosphoramide (Beaucage et al. (1993) Tetrahedron 49:1925)and references therein; Letsinger (1970) J. Org. Chem. 35:3800; Sprinzlet al. (1977) Eur. J. Biochem. 81: 579; Letsinger et al. (1986) Nucl.Acids Res. 14: 3487; Sawai et al. (1984) Chem. Lett. 805, Letsinger etal. (1988) J. Am. Chem. Soc. 110: 4470; and Pauwels et al. (1986)Chemica Scripta 26: 1419), phosphorothioate (Mag et al. (1991) NucleicAcids Res. 19:1437; and U.S. Pat. No. 5,644,048), phosphorodithioate(Briu et al. (1989) J. Am. Chem. Soc. 111 :2321, O-methylphophoroamiditelinkages (see Eckstein, Oligonucleotides and Analogues: A PracticalApproach, Oxford University Press), and peptide nucleic acid backbonesand linkages (see Egholm (1992) J. Am. Chem. Soc. 114:1895; Meier et al.(1992) Chem. Int. Ed. Engl. 31: 1008; Nielsen (1993) Nature, 365: 566;Carlsson et al. (1996) Nature 380: 207). Other analog nucleic acidsinclude those with positive backbones (Denpcy et al. (1995) Proc. Natl.Acad. Sci. USA 92: 6097; non-ionic backbones (U.S. Pat. Nos. 5,386,023,5,637,684, 5,602,240, 5,216,141 and 4,469,863; Angew. (1991) Chem. Intl.Ed. English 30: 423; Letsinger et al. (1988) J. Am. Chem. Soc. 110:4470;Letsinger et al. (1994) Nucleoside & Nucleotide 13:1597; Chapters 2 and3, ASC Symposium Series 580, “Carbohydrate Modifications in AntisenseResearch”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al. (1994),Bioorganic & Medicinal Chem. Lett. 4: 395; Jeffs et al. (1994) J.Biomolecular NMR 34:17; Tetrahedron Lett. 37:743 (1996) and non-ribosebackbones, including those described in U.S. Pat. Nos. 5,235,033 and5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, CarbohydrateModifications in Antisense Research, Ed. Y. S. Sanghui and P. Dan Cook.Nucleic acids containing one or more carbocyclic sugars are alsoincluded within the definition of nucleic acids (see Jenkins et al.(1995), Chem. Soc. Rev. pp169-176). Several nucleic acid analogs aredescribed in Rawls, C & E News Jun. 2, 1997 page 35. These modificationsof the ribose-phosphate backbone may be done to facilitate the additionof additional moieties such as labels, or to increase the stability andhalf-life of such molecules in physiological environments. As usedherein, oligonucleotide is substantially equivalent to the terms“amplimers”, “primers”, “oligomers”, and “probes”, as commonly definedin the art.

[0072] The term “heterologous” as it relates to nucleic acid sequencessuch as coding sequences and control sequences, denotes sequences thatare not normally associated with a region of a recombinant construct,and/or are not normally associated with a particular cell. Thus, a“heterologous” region of a nucleic acid construct is an identifiablesegment of nucleic acid within or attached to another nucleic acidmolecule that is not found in association with the other molecule innature. For example, a heterologous region of a construct could includea coding sequence flanked by sequences not found in association with thecoding sequence in nature. Another example of a heterologous codingsequence is a construct where the coding sequence itself is not found innature (e.g., synthetic sequences having codons different from thenative gene). Similarly, a host cell transformed with a construct whichis not normally present in the host cell would be consideredheterologous for purposes of this invention.

[0073] A “coding sequence” or a sequence which “encodes” a particularpolypeptide (e.g. a methyltransferase, etc.), is a nucleic acid sequencewhich is ultimately transcribed and/or translated into that polypeptidein vitro and/or in vivo when placed under the control of appropriateregulatory sequences. In certain embodiments, the boundaries of thecoding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxy) terminus. Acoding sequence can include, but is not limited to, cDNA fromprocaryotic or eucaryotic mRNA, genomic DNA sequences from procaryoticor eucaryotic DNA, and even synthetic DNA sequences. In preferredembodiments, a transcription termination sequence will usually belocated 3′ to the coding sequence.

[0074] The term “ortholog” refers to genes or proteins which arehomologs via speciation, e.g., closely related and assumed to havecommon descent based on structural and functional considerations.Orthologous proteins function as recognizably the same activity indifferent species.

[0075] Expression “control sequences” or “regulatory elements” referscollectively to promoter sequences, ribosome binding sites,polyadenylation signals, transcription termination sequences, upstreamregulatory domains, enhancers, and the like, which collectively providefor the transcription and translation of a coding sequence in a hostcell. Not all of these control sequences need always be present in arecombinant vector so long as the desired gene is capable of beingtranscribed and translated.

[0076] “Recombination” refers to the reassortment of sections of DNA orRNA sequences between two DNA or RNA molecules. “Homologousrecombination” occurs between two DNA molecules which hybridize byvirtue of homologous or complementary nucleotide sequences present ineach DNA molecule.

[0077] The terms “stringent conditions” or “hybridization understringent conditions” refers to conditions under which a probe willhybridize preferentially to its target subsequence, and to a lesserextent to, or not at all to, other sequences. “Stringent hybridization”and “stringent hybridization wash conditions” in the context of nucleicacid hybridization experiments such as Southern and northernhybridizations are sequence dependent, and are different under differentenvironmental parameters. An extensive guide to the hybridization ofnucleic acids is found in Tij ssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes part I chapter 2 Overview of principles of hybridization and thestrategy of nucleic acid probe assays, Elsevier, New York. Generally,highly stringent hybridization and wash conditions are selected to beabout 5° C. lower than the thermal melting point (T_(m)) for thespecific sequence at a defined ionic strength and pH. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Very stringentconditions are selected to be equal to the T_(m) for a particular probe.

[0078] An example of stringent hybridization conditions forhybridization of complementary nucleic acids which have more than 100complementary residues on a filter in a Southern or northern blot is 50%formamide with 1 mg of heparin at 42° C., with the hybridization beingcarried out overnight. An example of highly stringent wash conditions is0.15 M NaCl at 72° C. for about 15 minutes. An example of stringent washconditions is a 0.2×SSC wash at 65° C. for 15 minutes (see, Sambrook etal. (1989) Molecular Cloning—A Laboratory Manual (2nd ed.) Vol. 1-3,Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, for adescription of SSC buffer). Often, a high stringency wash is preceded bya low stringency wash to remove background probe signal. An examplemedium stringency wash for a duplex of, e.g., more than 100 nucleotides,is 1×SSC at 45° C. for 15 minutes. An example low stringency wash for aduplex of, e.g., more than 100 nucleotides, is 4-6×SSC at 40° C. for 15minutes. In general, a signal to noise ratio of 2× (or higher) than thatobserved for an unrelated probe in the particular hybridization assayindicates detection of a specific hybridization. Nucleic acids which donot hybridize to each other under stringent conditions are stillsubstantially identical if the polypeptides which they encode aresubstantially identical. This occurs, e.g., when a copy of a nucleicacid is created using the maximum codon degeneracy permitted by thegenetic code.

[0079] “Expression vectors” are defined herein as nucleic acid sequencesthat are direct the transcription of cloned copies of genes/cDNAs and/orthe translation of their mRNAs in an appropriate host. Such vectors canbe used to express genes or cDNAs in a variety of hosts such asbacteria, bluegreen algae, plant cells, insect cells and animal cells.Expression vectors include, but are not limited to, cloning vectors,modified cloning vectors, specifically designed plasmids or viruses.Specifically designed vectors allow the shuttling of DNA between hosts,such as bacteria-yeast or bacteria-animal cells. An appropriatelyconstructed expression vector preferably contains: an origin ofreplication for autonomous replication in a host cell, a selectablemarker, optionally one or more restriction enzyme sites, optionally oneor more constitutive or inducible promoters. In preferred embodiments,an expression vector is a replicable DNA construct in which a DNAsequence encoding LaeA or a fragment thereof is operably linked tosuitable control sequences capable of effecting the expression of theproducts in a suitable host. Control sequences include a transcriptionalpromoter, an optional operator sequence to control transcription andsequences which control the termination of transcription andtranslation, and so forth.

[0080] A “polymorphism” is a variation in the DNA sequence of somemembers of a species. A polymorphism is thus said to be “allelic,” inthat, due to the existence of the polymorphism, some members of aspecies may have the unmutated sequence (i.e. the original “allele”)whereas other members may have a mutated sequence (i.e. the variant ormutant “allele”). In the simplest case, only one mutated sequence mayexist, and the polymorphism is said to be diallelic. In the case ofdiallelic diploid organisms, three genotypes are possible. They can behomozygous for one allele, homozygous for the other allele orheterozygous. In the case of diallelic haploid organisms, they can haveone allele or the other, thus only two genotypes are possible. Theoccurrence of alternative mutations can give rise to trialleleic, etc.polymorphisms. An allele may be referred to by the nucleotide(s) thatcomprise the mutation.

[0081] “Single nucleotide polymorphism” or “SNPs are defined by theircharacteristic attributes. A central attribute of such a polymorphism isthat it contains a polymorphic site, “X,” most preferably occupied by asingle nucleotide, which is the site of the polymorphism's variation(Goelet and Knapp U.S. patent application Ser. No. 08/145,145). Methodsof identifying SNPs are well known to those of skill in the art (see,e.g., U.S. Pat. No. 5,952,174).

[0082] Abbreviations used herein include aa, amino acid; MMG, minimalmedia glucose; MMT, minimal media threonine; OE, over expression; LB,Luria-Bertani; nt, nucleotide; ORF, open reading frame; PCR, polymerasechain reaction; PEG, polyethyleneglycol; R, resistant; WT, wild-type;and TS, temperature sensitive.

[0083] III. The Invention

[0084] The invention is based on the discovery of a new fungal gene andprotein encoded thereby which regulates the activity of multiplesecondary metabolite gene clusters in fungi. Appropriate expression ofthe gene, laeA, provides increased production of secondary metabolitesin engineered cells. In particular, such a method of increasingsecondary metabolite allows the production of improved yields ofvaluable products including, but not limited to, lovastatin andpenicillin.

[0085] Nucleic acids encoding LaeA were first identified by the presentinventors in a screen designed to decouple asexual sporulation from STbiosynthesis in a norsolorinic acid (NOR) accumulating strain of A.nidulans. Various mutants displaying a phenotype of normal sporulationcombined with loss of ST production were genetically characterized andplaced into two groups depending on genetic linkage to the ST cluster.The non-linked mutants were further characterized with regard to theirability to regulate aflR transcription. Several of these mutants werecomplemented by an A. nidulans cosmid library. The complementation ofaflR subsequently yielded the gene, laeA, which appears to encode anovel protein methyltransferase. Upon functional characterization, thelaeA gene was discovered to possess characteristics of a global, oruniversal, regulator of secondary metabolism that is required not onlyfor ST biosynthesis and mycelial mat pigmentation but also for PN andLOV biosynthesis.

[0086] In one embodiment, the invention is directed to the polypeptideset forth in SEQ ID NO:3 (i.e., the mature polypeptide) as well aspolypeptides and fragments, particularly those which have the biologicalactivity of LaeA, and also those which have at least 85% identity overtheir length to a polypeptide of SEQ ID NO:3, and more preferably atleast 90% identity over their length to a polypeptide of SEQ ID NO:3,and still more preferably at least 95% identity over their length to apolypeptide of SEQ ID NO:3.

[0087] A polypeptide fragment according to the invention is apolypeptide having an amino acid sequence that is entirely the same aspart but not all of the amino acid sequence of the aforementionedpolypeptides. LaeA polypeptide fragments may be “free-standing,” orcomprised within a larger polypeptide of which they form a part orregion, most preferably as a single continuous region, a single largerpolypeptide.

[0088] Preferred fragments include, for example, truncation polypeptideshaving a portion of an amino acid sequence of SEQ ID NO:3, or ofvariants thereof, such as a continuous series of residues that includesthe amino terminus, or a continuous series of residues that includes thecarboxyl terminus. Further preferred are fragments characterized bystructural or functional attributes such as fragments that comprisecatalytic domains, alpha-helix and alpha-helix forming regions,beta-sheet and beta-sheet-forming regions, turn and turn-formingregions, coil and coil-forming regions, hydrophilic regions, hydrophobicregions, alpha amphipathic regions, beta amphipathic regions, flexibleregions, surface-forming regions, substrate binding region, and highantigenic index regions.

[0089] Also preferred are biologically active fragments which are thosefragments that mediate activities of LaeA, including those with asimilar activity or an improved activity, or with a decreasedundesirable activity. Particularly preferred fragments are those capableof global regulation of secondary metabolite biosynthesis. Also includedare those fragments that are antigenic or immunogenic in an animal,especially in a human. Fragments of the polypeptides of the inventionmay be employed for producing the corresponding full-length polypeptideby peptide synthesis; therefore, these particular fragments may beemployed as intermediates for producing the full-length polypeptides ofthe invention.

[0090] The present invention also encompasses nucleic acids which encodeLaeA. Accordingly, any nucleic acid sequence which encodes the aminoacid sequence of LaeA can be used to produce recombinant molecules whichexpress LaeA. In a particular embodiment, the invention encompasses thenucleic acid comprising the nucleic acid sequence of SEQ ID NO:1 or SEQID NO:2.

[0091] It will be appreciated by those skilled in the art that as aresult of the degeneracy of the genetic code, a multitude of nucleotidesequences encoding LaeA, some bearing minimal homology to the nucleotidesequences of any known and naturally occurring gene, may be produced.Thus, the invention contemplates each and every possible variation ofnucleotide sequence that could be made by selecting combinations basedon possible codon choices. These combinations are made in accordancewith the standard triplet genetic code as applied to the nucleotidesequence of naturally occurring LaeA, and all such variations are to beconsidered as being specifically disclosed.

[0092] Preferred embodiments of the invention are polynucleotides thatare at least 70% identical over their entire length to a polynucleotideset out in SEQ ID NO:2, and polynucleotides that are complementary tothe same. Alternatively, most highly preferred are polynucleotides thatcomprise a region that is at least 80% identical over its entire lengthto a polynucleotide set out in SEQ ID NO:2 and polynucleotidescomplementary thereto. In this regard, polynucleotides at least 90%identical over their entire length to the same are particularlypreferred, and among these particularly preferred polynucleotides, thosewith at least 95% are especially preferred. Furthermore, those with atleast 97% are highly preferred, and among these those with at least 98%and at least 99% are particularly highly preferred, with at least 99%being the more preferred.

[0093] Although nucleotide sequences which encode LaeA and its variantsare preferably capable of hybridizing to the nucleotide sequence of thenaturally occurring LaeA under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding LaeA or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding LaeA and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

[0094] The invention also encompasses production of DNA sequences, orfragments thereof, which encode LaeA and its derivatives, entirely bysynthetic chemistry. After production, the synthetic sequence may beinserted into any of the many available expression vectors and cellsystems using reagents that are well known in the art. Moreover,synthetic chemistry may be used to introduce mutations into a sequenceencoding LaeA or any fragment thereof.

[0095] Also encompassed by the invention are nucleotide sequences thatare capable of hybridizing to the claimed nucleic acids, and inparticular, those that encode the amino acid sequence set forth in SEQID NO:3, under various conditions of stringency as taught in Wahl, G. M.and S. L. Berger (1987; Methods Enzymol. 152:399-407) and Kimmel, A. R.(1987; Methods Enzymol. 152:507-511), preferably highly stringenthybridization conditions, as defined herein.

[0096] Methods for DNA sequencing which are well known and generallyavailable in the art and may be used to practice any of the embodimentsof the invention. The methods may employ such enzymes as the Klenowfragment of DNA polymerase I, SEQUENASE.RTM. (US Biochemical Corp,Cleveland, Ohio), Taq polymerase (Perkin Elmer), thermostable T7polymerase (Amersham, Chicago, Ill.), or combinations of polymerases andproofreading exonucleases such as those found in the ELONGASEAmplification System marketed by Gibco/BRL (Gaithersburg, Md.).Preferably, the process is automated with machines such as the HamiltonMicro Lab 2200 (Hamilton, Reno, Nev.), Peltier Thermal Cycler (PTC200;MJ Research, Watertown, Me.) and the ABI Catalyst and 373 and 377 DNASequencers (Perkin Elmer).

[0097] The nucleic acid sequences encoding LaeA may be extendedutilizing a partial nucleotide sequence and employing various methodsknown in the art to detect upstream sequences such as promoters andregulatory elements. For example, one method which may be employed,“restriction-site” PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322). In particular, genomic DNA is first amplified in thepresence of primer to a linker sequence and a primer specific to theknown region. The amplified sequences are then subjected to a secondround of PCR with the same linker primer and another specific primerinternal to the first one. Products of each round of PCR are transcribedwith an appropriate RNA polymerase and sequenced using reversetranscriptase.

[0098] Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186). The primers may be designed usingcommercially available software such as OLIGO 4.06 Primer Analysissoftware (National Biosciences Inc., Plymouth, Minn.), or anotherappropriate program, to be 22-30 nucleotides in length, to have a GCcontent of 50% or more, and to anneal to the target sequence attemperatures about 68.degree.-72.degree C. The method uses severalrestriction enzymes to generate a suitable fragment in the known regionof a gene. The fragment is then circularized by intramolecular ligationand used as a PCR template.

[0099] Another method which may be used is capture PCR which involvesPCR amplification of DNA fragments adjacent to a known sequence in humanand yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1:111-119). In this method, multiple restriction enzymedigestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown fragment of the DNA moleculebefore performing PCR. Another method which may be used to retrieveunknown sequences is that of Parker, J. D. et al. (1991; Nucleic AcidsRes. 19:3055-3060). Additionally, one may use PCR, nested primers, andPROMOTER FINDER.TM. libraries to walk genomic DNA (Clontech, Palo Alto,Calif.). This process avoids the need to screen libraries and is usefulin finding intron/exon junctions.

[0100] When screening for full-length cDNAs, it is preferable to uselibraries that have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable, in that they will contain moresequences which contain the 5′ regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into 5′ non-transcribedregulatory regions.

[0101] Capillary electrophoresis systems which are commerciallyavailable may be used to analyze the size or confirm the nucleotidesequence of sequencing or PCR products. In particular, capillarysequencing may employ flowable polymers for electrophoretic separation,four different fluorescent dyes (one for each nucleotide) which arelaser activated, and detection of the emitted wavelengths by a chargecoupled device camera. Output/light intensity may be converted toelectrical signal using appropriate software (e.g. GENOTYPER.TM. andSEQUENCE NAVIGATOR.TM., Perkin Elmer) and the entire process fromloading of samples to computer analysis and electronic data display maybe computer controlled. Capillary electrophoresis is especiallypreferable for the sequencing of small pieces of DNA which might bepresent in limited amounts in a particular sample.

[0102] In another embodiment of the invention, nucleotide sequences orfragments thereof which encode LaeA may be used in recombinant DNAmolecules to direct expression of LaeA, fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced, and these sequences may be used to clone and expressLaeA.

[0103] As will be understood by those of skill in the art, it may beadvantageous to produce LaeA-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce an RNA transcript havingdesirable properties, such as a half-life which is longer than that of atranscript generated from the naturally occurring sequence.

[0104] The nucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterLaeA-encoding sequences for a variety of reasons, including but notlimited to, alterations which modify the cloning, processing, and/orexpression of the gene product. DNA shuffling by random fragmentationand PCR reassembly of gene fragments and synthetic oligonucleotides maybe used to engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, introduce mutations, and so forth.

[0105] In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding LaeA may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of LaeA activity, it may be useful toencode a chimeric LaeA protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the LaeA encoding sequence and theheterologous protein sequence, so that LaeA may be cleaved and purifiedaway from the heterologous moiety.

[0106] In another embodiment, sequences encoding LaeA may besynthesized, in whole or in part, using chemical methods well known inthe art (see Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser.215-223, Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of LaeA, or a fragment thereof.For example, peptide synthesis can be performed using varioussolid-phase techniques (Roberge, J. Y. et al. (1995) Science269:202-204) and automated synthesis may be achieved, for example, usingthe ABI 431A Peptide Synthesizer (Perkin Elmer).

[0107] The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Structures and Molecular Principles, W H Freeman andCo., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, supra). Additionally, the amino acidsequence of LaeA, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

[0108] In order to express a biologically active LaeA, the nucleotidesequences encoding LaeA or functional equivalents, may be inserted intoappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence. Methods which are well known to those skilled in theart may be used to construct expression vectors containing sequencesencoding LaeA and appropriate transcriptional and translational controlelements. These methods include in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. Such techniquesare described in Sambrook, J. et al. (1989) Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., andAusubel, F. M. et al. (1989) Current Protocols in Molecular Biology,John Wiley & Sons, New York, N.Y.

[0109] A variety of expression vector/host systems may be utilized tocontain and express sequences encoding LaeA. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Theinvention is not limited by the host cell employed.

[0110] The “control elements” or “regulatory sequences” are thosenon-translated regions of the vector—enhancers, promoters, 5′ and 3′untranslated regions—which interact with host cellular proteins to carryout transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or PSPORT1 plasmid (Gibco BRL) and the like may be used.The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding LaeA,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

[0111] In bacterial systems, a number of expression vectors may beselected depending upon the use intended for LaeA. For example, whenlarge quantities of LaeA are needed for the induction of antibodies,vectors which direct high level expression of fusion proteins that arereadily purified may be used. Such vectors include, but are not limitedto, the multifunctional E. coli cloning and expression vectors such asBLUESCRIPT.RTM. (Stratagene), in which the sequence encoding LaeA may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of .beta.-galactosidase so that ahybrid protein is produced; pIN vectors (Van Heeke, G. and S. M.Schuster (1989) J. Biol. Chem. 264:5503-5509); and the like. pGEXvectors (Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

[0112] In the yeast, Saccharomyces cerevisiae, a number of vectorscontaining constitutive or inducible promoters such as alpha factor,alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al.(supra) and Grant et al. (1987) Methods Enzymol. 153:516-544.

[0113] In cases where plant expression vectors are used, the expressionof sequences encoding LaeA may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J.3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter,J. et al. (1991) Results Probl. Cell Differ. 17:85-105). Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. Such techniques aredescribed in a number of generally available reviews (see, for example,Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York, N.Y.; pp. 191-196).

[0114] An insect system may also be used to express LaeA. For example,in one such system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes in Spodopterafrugiperda cells or in Trichoplusia larvae. The sequences encoding LaeAmay be cloned into a non-essential region of the virus, such as thepolyhedrin gene, and placed under control of the polyhedrin promoter.Successful insertion of LaeA will render the polyhedrin gene inactiveand produce recombinant virus lacking coat protein. The recombinantviruses may then be used to infect, for example, S. frugiperda cells orTrichoplusia larvae in which LaeA may be expressed (Engelhard, E. K. etal. (1994) Proc. Nat. Acad. Sci. 91:3224-3227).

[0115] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, sequences encoding LaeA may be ligated into anadenovirus transcription/translation complex consisting of the latepromoter and tripartite leader sequence. Insertion in a non-essential E1or E3 region of the viral genome may be used to obtain a viable viruswhich is capable of expressing LaeA in infected host cells (Logan, J.and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

[0116] Specific initiation signals may also be used to achieve moreefficient translation of sequences encoding LaeA. Such signals includethe ATG initiation codon and adjacent sequences. In cases wheresequences encoding LaeA, its initiation codon, and upstream sequencesare inserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a fragment thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers which are appropriate for theparticular cell system which is used, such as those described in theliterature (Scharf, D. et al. (1994) Results Probl. Cell Differ.20:125-162).

[0117] In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells which have specific cellular machineryand characteristic mechanisms for post-translational activities (e.g.,CHO, HeLa, MDCK, HEK293, and WI38), are available from the American TypeCulture Collection (ATCC; Bethesda, Md.) and may be chosen to ensure thecorrect modification and processing of the foreign protein.

[0118] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress LaeA may be transformed using expression vectors which maycontain viral origins of replication and/or endogenous expressionelements and a selectable marker gene on the same or on a separatevector. Following the introduction of the vector, cells may be allowedto grow in an enriched media before they are switched to selectivemedia. The purpose of the selectable marker is to confer resistance toselection, and its presence allows growth and recovery of cells whichsuccessfully express the introduced sequences. Resistant clones ofstably transformed cells may be proliferated using culture techniquesappropriate to the cell type.

[0119] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1980)Cell 22:817-23) genes which can be employed in tk.sup.- oraprt.sup.-cells, respectively. Also, antimetabolite, antibiotic orherbicide resistance can be used as the basis for selection; forexample, dhfr which confers resistance to methotrexate (Wigler, M. etal. (1980) Proc. Natl. Acad. Sci. 77:3567-70); npt, which confersresistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin,F. et al (1981) J. Mol. Biol. 150:1-14) and als or pat, which conferresistance to chlorsulfuron and phosphinotricin acetyltransferase,respectively (Murry, supra). Additional selectable genes have beendescribed, for example, trpB, which allows cells to utilize indole inplace of tryptophan, or hisD, which allows cells to utilize histinol inplace of histidine (Hartman, S.C. and R. C. Mulligan (1988) Proc. Natl.Acad. Sci. 85:8047-51). Recently, the use of visible markers has gainedpopularity with such markers as anthocyanins, .beta glucuronidase andits substrate GUS, and luciferase and its substrate luciferin, beingwidely used not only to identify transformants, but also to quantify theamount of transient or stable protein expression attributable to aspecific vector system (Rhodes, C. A. et al. (1995) Methods Mol. Biol.55:121-131).

[0120] Although the presence/absence of marker gene expression suggeststhat the gene of interest is also present, its presence and expressionmay need to be confirmed. For example, if the sequence encoding LaeA isinserted within a marker gene sequence, transformed cells containingsequences encoding LaeA can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with asequence encoding LaeA under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

[0121] Alternatively, host cells which contain the nucleic acid sequenceencoding LaeA and express LaeA may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein.

[0122] The presence of polynucleotide sequences encoding LaeA can bedetected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes or fragments or fragments of polynucleotides encoding LaeA.Nucleic acid amplification based assays involve the use ofoligonucleotides or oligomers based on the sequences encoding LaeA todetect transformants containing DNA or RNA encoding LaeA.

[0123] A variety of protocols for detecting and measuring the expressionof LaeA, using either polyclonal or monoclonal antibodies specific forthe protein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson LaeA is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

[0124] A wide variety of labels and conjugation techniques are known bythose skilled in the art and may be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides encoding LaeAinclude oligolabeling, nick translation, end-labeling or PCRamplification using a labeled nucleotide. Alternatively, the sequencesencoding LaeA, or any fragments thereof may be cloned into a vector forthe production of an mRNA probe. Such vectors are known in the art, arecommercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase such as T7, T3, orSP6 and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo,Mich.); Promega (Madison Wis.); and U.S. Biochemical Corp., (Cleveland,Ohio)). Suitable reporter molecules or labels, which may be used forease of detection, include radionuclides, enzymes, fluorescent,chemiluminescent, or chromogenic agents as well as substrates,cofactors, inhibitors, magnetic particles, and the like

[0125] Host cells transformed with nucleotide sequences encoding LaeAmay be cultured under conditions suitable for the expression andrecovery of the protein from cell culture. The protein produced by atransformed cell may be secreted or contained intracellularly dependingon the sequence and/or the vector used. As will be understood by thoseof skill in the art, expression vectors containing polynucleotides whichencode LaeA may be designed to contain signal sequences which directsecretion of LaeA through a prokaryotic or eukaryotic cell membrane.Other constructions may be used to join sequences encoding LaeA tonucleotide sequence encoding a polypeptide domain which will facilitatepurification of soluble proteins Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen, San Diego, Calif.) between the purificationdomain and LaeA may be used to facilitate purification. One suchexpression vector provides for expression of a fusion protein containingLaeA and a nucleic acid encoding 6 histidine residues preceding athioredoxin or an enterokinase cleavage site. The histidine residuesfacilitate purification on IMAC (immobilized metal ion affinityChromatography as described in Porath, J. et al. (1992, Prot. Exp.Purif. 3:263-281) while the enterokinase cleavage site provides a meansfor purifying LaeA from the fusion protein. A discussion of vectorswhich contain fusion proteins is provided in Kroll, D. J. et al. (1993;DNA Cell Biol. 12:441-453).

[0126] In addition to recombinant production, fragments of LaeA may beproduced by direct peptide synthesis using solid-phase techniques(Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesismay be performed using manual techniques or by automation. Automatedsynthesis may be achieved, for example, using Applied Biosystems 431APeptide Synthesizer (Perkin Elmer). Various fragments of LaeA may bechemically-synthesized separately and combined using chemical methods toproduce the full length molecule.

[0127] In another embodiment, the present invention is a method ofincreasing the production of a secondary metabolite in a secondarymetabolite-producing organism. Based upon the foregoing, one of skill inthe art may transform a secondary metabolite-producing organism with anucleic acid as described above as well as expression vectors comprisingsuch nucleic acids. The nucleic acid is preferably over expressedfollowing the transformation step resulting in an increase in secondarymetabolite production in the transformed organism. The increase insecondary metabolite production is greater than 2 fold as compared to anuntransformed control. The inventors have observed 400-900% increases inpenicillin production in A. nidulans as well as 500-700% increases oflovastin in A. terreus. In certain embodiments, the secondary metabolitebenefiting from increased production is lovastin, sterigmatocystin,penicillin, or gliotoxin. Preferred secondary metabolite-producingorganisms include Aspergillus species, preferably A. nidulans or A.terreus or A. fumigatus. A detailed example of how such a method ofincreasing the production of a secondary metabolite may be carried outis described below in the EXAMPLES section.

[0128] In another embodiment directed to improving yields of secondarymetabolites from host systems, the method according to the invention mayutilize a non-secondary metabolite producing organism and include theadditional step of transforming said organism with an entirebiosynthetic gene cluster or at least biosynthetic genes sufficient forthe production of a secondary metabolite or a secondary metabolitederivative or analog. The additional expression, preferablyoverexpression, of LaeA in the transformed organism may then enhance theproduction of a desired secondary metabolite.

[0129] In other embodiments, the secondary metabolite benefiting fromincreased production is a rare or minor secondary metabolite speciesthat, without the amplification provided by the present invention, wouldnot be present in amounts allowing identification and/or purification.Following amplification based on the present invention, dentificationand purification of rare or minor species may, of course, be carried outby techniques known to the skilled artisan such as, for example, thinlayer chromatography (TLC) followed by mass spectrometry (MS). Thus, itcan be appreciated that the present invention provides a new andadvantageous key to allow enhanced screening and identification of newand useful secondary metabolites.

[0130] In yet other embodiments of the invention, overexpression and/ordeletion laeA strains may be utilized in novel screens for new anduseful secondary metabolite biosynthesis gene clusters. For example,using standard microarray technology now commonly employed in the field,one of skill in the art may construct a microarray containing, forexample, nucleic acids representative of the expressed genes ofwild-type A. nidulans (see, for example, D. Bowtell and J. Sambrook, DNAMicroarrays: A Molecular Cloning Manual (2000) Cold Spring HarborLaboratory Press and P. Baldi and G. W. Hatfield, DNA Microarrays andGene Expression: From Experiments to Data Analysis and Modeling (2002)Cambridge University Press describing standard microarray techniquesdata analyses applicable in the present invention). The entire genomefor A. nidulans has been sequenced and the sequence is available inannotated form for public use (see the Whitehead Institute/MIT Centerfor Genome Research websitehttp://www-genome.wi.mit.edu/annotation/funi/aspergillus/). Constructionof the specific nucleic acids affixed to the array substrate may bebased on, for example, an expressed sequence tag database provided bythe University of Oklahoma (see http://www.genome.ou.edu.fungal.html).Using the microarray and standard hybridization techniques known in thefield, the expression levels of genes in wild-type A. nidulans versus anlaeA deletion mutant may then be compared to identify genes whoseexpression is reduced or absent in the laeA deletion mutant compared tothe wild-type line. The artisan may subsequently examine the genomicsequence available for A. nidulans in order to identify putativesecondary metabolite biosynthesis cluster genes in the immediatevicinity of the relevant gene whose expression is initially identifiedas affected by the absence of laeA expression. As secondary metabolitebiosynthesis genes are well known to occur in clustered fashion, asdescribed in a plurality of references cited herein, new putativesecondary metabolite gene clusters may be identified by this approach.Further, genes within a putative gene cluster may subsequently bedisrupted and the mutant line's production of secondary metaboliteproducts may then be compared with wild-type production in plus/minusfashion to identify the specific natural product produced by thenewly-identified gene cluster. The natural product may then be isolatedand characterized using standard techniques described and referencedherein.

[0131] It is envisioned the above-described screening strategies may becarried out not only between wild-type and laeA deletion mutants butalso, and more preferably, between laeA overexpression mutants and laeAdeletion mutants to obain the greatest contrast in laeA-influencedsecondary metabolite biosynthesis gene expression. As well, thescreening methodology described herein is not limited to any oneparticular fungus but may be applied to any fungus having an laeAortholog (e.g., Aspergillus other than A. nidulans). For example, thegenome for Fusarium graminearum is now available and screens utilizinglaeA overexpression or disruption strains to identify new F. graminearumsecondary metabolite gene clusters may certainly be carried out based onthe novel materials and teachings provided herein (also see WhiteheadInstitute/MIT Center for Genomic Research website athttp://www-genome.wi.mit.edu/annotation/-fungi/fgi/ for F. graminearumgenomic sequence).

[0132] In order to demonstrate the utility of the above-describedscreening methodology, the inventors carried out differential geneexpression analyses with a microarray using unigene sequences availablefrom an A. nidulans expressed sequence tag database (available throughtthe University of Oklahoma websitehttp://www.genome.ou.edu.fungal.html). In addition, approximately 145gene sequences from GenBank that are not represented in the EST databasewere included, as well as the sequence for laeA (provided herein) Theresulting array represented 6,529 unique gene sequences; this is asubstantial portion of the approximately 10,000 expressed genes of theA. nidulans genome. Probe sequences of 24 base pairs were created andestimated to provide approximately 14 sequences per gene. Thesesequences were synthesized on chips by Nimblegen, Inc. (Madison, Wis.),using proprietary maskless technology. Total RNA was prepared from FGSC26 (biA1; veA1) and RJW40.7 (biA1; ΔlaeA::metG;veA1) using TRIzol®reagent (Invitrogen, Carlsbad, Calif.) followed by RNeasy clean up(Qiagen Inc., Valencia, Calif.). The fungal strains used for thisprocedure are further detailed in Table 1 below. Total RNA was spikedwith control RNA transcripts, converted to biotinylated cRNA andfragmented following the Affymetrix Expression Analysis Technical Manual(rev 1). Hybridization mixtures were prepared according to the arraymanufacturer's standard protocol using 10 mg biotinylated cRNA. Sampleswere incubated with the arrays overnight at 42° C. Chips were washed,stained with streptavadin-linked Cy3 dye, and dried according to themanufacturer's protocol. Chips were scanned using a GenePix scanner(Axon Instruments, Union City, Calif.). The data were converted to aMicrosoft Access database and normalized by the RNA spike controlsignals. Genes dependent on LaeA for expression were determined byexpression ratios (wild type to mutant deleted laeA strain). Among thelowests ratios were genes known to be involved in penicillin andsterigmatocystin biosynthesis, consistent with other experimental data.Two additional LaeA-dependent genes were found to be adjacent to eachother in the A. nidulans genome sequence (now annotated as AN8439.1 andAN8440.1). These genes are within 10 kb of genes encoding anon-ribosomal peptide synthase (AN8433.1), a tyrosinase (AN8435.1), anda P450 monooxygenase (AN8437.1), enzymes that are hallmarks of secondarymetabolic pathways. Thus, the method according to the present inventionwas useful in the initial step of identifying a putative secondarymetabolite biosynthesis gene cluster. This putative cluster is nowavailable to be further characterized and defined using standardmethodologies.

[0133] The present invention is also a method of inhibiting or reducingproduction of a secondary metabolite by replacement of thenaturally-occurring laeA or a laeA homologue with a polynucleotideencoding a variant of the polypeptide as set forth in SEQ ID NO: 3wherein the variant is altered by mutagenesis or equivalent technique tobe nonfunctional in terms of increasing or regulating secondarymetabolite production. Such a gene replacement exercise could be carriedout by one of skill in the art using techniques presently known in thefield. Such a method would be useful in reducing or eliminatingproduction of toxic secondary metabolites in certain organisms. Forexample, a non-functional variant of laeA would be useful in reducing oreliminating aflatoxin production in an A. parasiticus or A. flavusstrain transformed thereby (e.g., the ΔlaeA strains described in thefollowing section are illustrative of this method). In addition, LaeAmay be targeted by a therapeutic such that LaeA's ability to regulatesecondary metabolite gene cluster activity is inhibited. This approachwould provide a therapeutic able to reduce the virulence of cells ororganisms thereby providing a treatment for medical maladies involvingfungal infections. Methods of identifying inhibitors of target moleculesare well known in the art.

[0134] In another embodiment, the present invention includes thetransformed organisms described above. These organisms include secondarymetabolite-producing organisms, preferably yeast and fungi, that havebeen engineered to display at least a 2 fold increase in secondarymetabolite production, preferably where the secondary metabolites arelovastatin or pencillin. These organisms also include non-secondarymetabolite-producing organisms, preferably yeast or fungi, that havebeen engineered to produce secondary metabolites. Transformed organismsmay comprise a nucleic acid described above as well as expressionvectors including such a nucleic acid.

[0135] The following examples are offered for illustrative purposesonly, and are not intended to limit the scope of the present inventionin any way. Indeed, various modifications of the invention in additionto those shown and described herein will become apparent to thoseskilled in the art from the foregoing description and fall within thescope of the appended claims.

IV. EXAMPLES

[0136] A. Materials and Methods

[0137] Fungal Strains and Growth Conditions

[0138] Table 1 lists all fungal strains used herein. Some strains arenot discussed in the text but were used for sexual crosses to obtain thestrains of interest. Sexual crosses of A. nidulans strains wereconducted according to Pontecorvo et al.²⁶ All strains were maintainedas glycerol stocks and were grown at 37° C. for A. nidulans and A.fumigatus or 32° C. for A. terreus on glucose minimal medium (GMM)⁴⁶,threonine minimal medium (TMM)¹⁶ or lactose minimal medium (LMM)⁹amended with 30 mM cyclopentanone. Threonine and cyclopentanone bothinduce alcA(p) which was used to promote laeA expression. All mediacontained appropriate supplements to maintain auxotrophs²⁷. TABLE 2Fungal strains. Fungal Strains Genotype Source Wild type and controlsAspergillus nidulans strains FGSC 26 biAl; veAl FGSC^(a) RDIT 2.1 methGlD. Tsitsigiannis RAMB38 biAl; methGl; ΔaflR::argB, trpC801, veAl A. M.Bergh RDIT 2.3 veAl D. Tsitsigiannis RDIT 7.24 methGl; veAl D.Tsitsigiannis RDIT 30.34 methGl; trpC801; pyrG89, veAl D. TsitsigiannisRJH26 biAl; wA3; argB2; ΔstcE::argB, veAl, trpC801 this study RJW3pyrG89; wA3, argB2; pyroA4, ΔstcE::argB, veAl, trpC801 this study RJW51alcA(p)::lovB::pyr4; hygromycinB::lov gene cluster this study RKIS 1pabaAl, yA2; veAl ³⁹ RMFV2 pabaAl, yA2; veAl, argB2; ΔaflR::argB ⁴⁰TJH3.40 biAl; wA3; argB2; methGl; ΔstcE::argB2, veAl ⁴¹ TJH34.10 pabaAl,yA2; veAl, alcA(p)::aflR::trpC ⁴² TPK1.1 biAl; methGl; veAl N. KellerWMH1739 pabaAl, yA2, alcA(p)::lovB::pyr4; ⁴³ hygromycinB::lov genecluster A. fumigatus strains AF293 G. May AF293.1 pyrG⁻ G. May TJW55.2pyrG⁻, A. parasiticus pyrG this study A. terreus strains ATCC 20542ATCC^(b) TJW58.9 hygB this study laeA mutants Aspergillus nidulansstrains MRB300 biAl; wA3; methGl; ΔstcE::argB2; veAl; laeAl ⁴¹ RJW32biAl, wA3; argB2; methGl; ΔstcE::argB, veAl, trpC801 this studyΔlaeA::methG RJW33.2 wA3; argB2; methGl; pyroA4; ΔstcE::argB, veAl, thisstudy trpC801; ΔlaeA::methG RJW 40.4 biAl; methGl; veAl; ΔlaeA::methGthis study RJW 40.7 biAl; ΔlaeA::methG, veAl this study RJW 44.2 biAl;methGl; alcA(p)::laeA::trpC, veAl; ΔlaeA::methG this study RJW 46.4methGl; veAl; ΔlaeA::methG this study RJW 54.8 methGl; aflR::trpC, veAl;ΔlaeA::methG this study RJW 55.8 methGl; aflR::trpC, ΔaflR::argB, veAl,ΔlaeA::methG this study RYJ 8 biAl; wA3; ΔstcE::argB, veAl, trpC801;laeAl this study RJW52 alcA(p)::laeA::trpC; alcA(p)::lovB::pyr4;hygromycinB::lov gene cluster this study RJW53 ΔlaeA::methG;alcA(p)::lovB::pyr4; hygromycinB::lov gene cluster this study TJW46.16biAl; wA3; argB2; methGl; ΔstcE::argB, veAl; alcA(p)::gfp::laeA::trpC;ΔlaeA::methG this study TJW57.9 wA3; argB2; methGl; pyroA4; ΔstcE::argB,veAl, this study aflR::trpC; ΔlaeA::methG A. fumigatus strain TJW54.2ΔlaeA::A. parasiticus pyrG; pyrG⁻ this study A. terreus strains TJW58.2hygB; alcA(p)::laeA this study TJW58.4 hygB; alcA(p)::laeA this studyTJW58.7 hygB; alcA(p)::laeA this study TJW58.8 hygB; alcA(p)::laeA thisstudy TJW58.14 hygB; alcA(p)::laeA this study Signal transductionmutants H1FAD4 biAl; veAl; fadA^(G42R) ⁴⁴ RKIS 11.1 pabaAl, yA2; veAl,argB2; ΔfadA::argB ⁴⁵ RKIS 28.5 pabaAl, yA2; veAl, alcA(p)::ras^(A17V)::argB ⁴⁵ TBN39.5 biAl; methGl; argB2; ΔflbA::argB; veAl ⁴⁶ TJH34.10pabaAl, yA2; trpC801, trpC::alcA::aflR, veAl J. Hicks TKIS 18.11 pabaAl,yA2; Δ argB::trpC; trpC801, veAl; ΔpkaA::argB ⁴⁵ TKIS20.1 pabaAl, yA2;veAl, alc(p)::pkaA::trpC ⁴⁵ TSRB1.38 biAl; methGl; argB2; ΔsfaD::argB;veAl 407

[0139] Cloning and Sequence of the A. nidulans and A. fumigatus laeAGenes

[0140] The A. nidulans aflR expression mutant, RYJ8 (derived fromMRB300, see Supplementary Information), was transformed with an A.nidulans genomic cosmid library. Norsolorinic acid (NOR) producingtransformants were purified and a cosmid, pCOSJW3, that complemented themutation was rescued from one transformant. NOR is a visible precursorin the ST biosynthetic pathway and commonly used as an indicator of STproduction⁵. pJW15, a 4.5 kb KpnI-EcoRI subclone of pCOSJW3 alsocomplemented the mutation and was sequenced using synthetic primers andABI PRISM DNA sequencing kit (PerkinElmer Life Science). The mutantallele, laeA1, was sequenced after subcloning a 3 kb PCR fragment fromRYJ8 genomic DNA amplified with primers LAE1 and LAE2 (see SupplementaryInformation) into Zero Blunt TOPO vector (Invitrogen Co.) to producepJW31. RACE technology using Gene Racer Kit (Invitrogen Co.) wasemployed to clone laeA cDNA according to manufacturer's instruction. Thecloned cDNA was then sequenced. The Institute for Genomic Research(TIGR) contains partial A. fumigatus genome sequence(http://www.tigr.org/tdb/e2k1/afu1/). A putative A. fumigatus laeAhomolog was obtained by blasting the A. fumigatus data with the A.nidulans laeA sequence.

[0141] Nucleic Acid Analysis

[0142] Extraction of DNA from fingi and bacteria, restriction enzymedigestion, gel electrophoresis, blotting, hybridization and probepreparation were performed by standard methods^(16, 28). Total RNA wasextracted from Aspergillus strains using Trizol reagent (Invitrogen Co.)according to the manufacturer's instructions. RNA blots were hybridizedwith a 0.7 kb SacII-KpnI fragment from pRB7 containing the stcU codingregion¹⁸, a 1.3 kb EcoRV-XhoI fragment from pJW19 containing the aflRcoding region, a 3 kb HindIII fragment from pJW45.4 containing the laeAcoding region, a 1.1 kb EcoRI-HindIII fragment from pUCHH(458)containing the ipnA coding region²⁹, a 5 kb BamHI fragment from pWHM1401containing the lovE coding region⁹, and a 1.3 kb PCR product frompWHM1263 containing the lovC coding region⁹. Also A. nidulans cosmidspW07H03, pL11C09 and pL24B03 were used as probes. pL11C09 contains mostof the ST gene cluster, whereas pW07H03 and pL24B03 primarily containgenes located upstream and downstream of the ST gene cluster,respectively⁸.

[0143] Fungal Transformation Procedures

[0144] Fungal transformation essentially followed that of Miller etal.³⁰ with the modification of embedding the protoplasts in top agar(0.75%) rather than spreading them by a glass rod on solid media.

[0145] Methyltransferase Bioassay

[0146] Nuclei of wild type and ΔlaeA strains were extracted fromcultures grown in GMM liquid media at 37° C., 300 rpm for 36 h. Themycelia were pulverized in liquid nitrogen with a buffer (1.0M sorbitol,10 mM Tris.HCl, pH 7.5) and then subjected to centrifugation (SorvallGSA rotor, 2500 rpm for 10 min). Supernatants were transferred to newtubes and centrifuged at 10,000 rpm for 15 min to isolate nuclei. Theisolated nuclei were collected in 1.5 ml centrifuge tubes and 200 μladenosyl-L-(methyl-3H) methionine [Amersham Pharmacia Biotech., 76.0Ci/mmol, 1 mCi/ml in dilute HCl (pH 2.0):ethanol 9: 1, v/v)] was addedto 1 ml. The reaction mixture was incubated 3 h at 30° C. and thennuclear protein was extracted using Trizol reagent (Invitrogen Co.)according to the manufacturer's instructions. Protein extracts (160 μg)were separated on a 10% SDS PAGE gel and the dried gel was exposed onX-ray film for one month.

[0147] Construction of Transformation Vectors and Strains

[0148] Plasmids were generated using standard techniques. Primers arelisted in Table 2. pfu Turbo (Stratagene Co.) was used for PCRreactions. The A. nidulans disruption plasmid pJW34 was constructed byligating a 1.2 kb DNA fragment upstream of the laeA start codon (primersLae1 and LA2) and a 1.2 kb DNA fragment downstream of the laeA stopcodon (primers LA3 and Lae2) to either side of the methG gene in thepUG11-41 vector³¹. The 5′ end PCR product and 3′ end PCR product wereinserted into the SacI site and HindIII site of pUG11-41 by blunt endligation, respectively. pJW34 was used to disrupt the laeA gene (ΔlaeA)in TJH3.40 to create TJW35.5. TJW35.5 was subsequently sexually crossedto RDIT2.1 to create RJW46.4. Plasmid pJW47.4 was constructed to overexpress laeA from the alcA promoter³². The 2.5 kb coding sequence oflaeA was amplified with primers OEF and OER and ligated into the HindIIIsite of pCN2 which contains the 5′ half of the trpC gene and the alcApromoter². This resulted in an alcA(p)::laeA fusion referred to asOE::laeA in text. pJW47.4 was used to transform RJW32 to tryptophanauxotrophy to yield the strain TJW44.39. TJW44.39 was subsequentlysexually crossed to RDIT2.1 to create RJW47.3. pJW47.4 and a hygromycynB(hygB) resistance gene containing plasmid pUCH2-8³³ were used forcotransformation to introduce the over expression laeA construct into A.terreus ATCC20542. Transformants were selected in hygromycin B (500μg/ml) containing medium and confirmed by PCR and Southernhybridization. Five transformants, TJW58.2, TJW58.4, TJW58.7, TJW58.8and TJW58.14 containing hygB and OE::laeA, were examined for LOVproduction and TJW58.9 containing hygB alone was used as a control(Table 1). pJW45.4, containing a wild type copy of the laeA gene, wasused to complement the ΔlaeA strain RJW33.2. pJW45.4 was created byligating the 3 kb laeA gene (primers MT1 and OER) into the HindIII siteof pSH96. pSH96 contains the 5′ half of the trpC gene³⁴. RJW33.2 is asexual progeny of a cross between TJW35.5 and RJW3. pJW45.4 was used totransform RJW33.2 to produce TJW42.7. TJW42.7 was crossed with RDIT7.24sexually to create RJW49.1. Plasmids pJW48 and pJW49 were created tovisualize LaeA by fusing the green fluorescent protein (sGFP)gene^(35,36) to the N-terminal and C-terminal of LaeA, respectively.pJW48 was made by ligating the 0.7 kb gfp gene (primers GF1 and GF2) to5′ end of the 2.5 kb encoding region of laeA gene (primers GF3 and OER)and then the ligated fragment was inserted into the pCN2 HindIII site toyield the alcA(p)::gfp::laeA chimera. pJW49 was constructed byconsecutively ligating a 2 kb laeA coding region (primers OEF and GFP2),a 0.7 kb gfp gene (primers GFP31 and GFP4), and a 0.5 kb laeAtermination cassette (primers GFP5 and OER) into the HindIII site inpCN2 to yield an alcA(p)::laeA::gfp::laeAterm chimera. pJW48 and pJW49were used to transform RJW32 to yield transformants TJW46.16 (5′ GFP)and TJW47.9 (3′ GFP) respectively. The A. fumigatus laeA gene disruptionvector, pJW58, was constructed by inserting a 0.9 kb DNA fragmentupstream of the laeA start cordon (primers FUM1 and FUM2), and a 1.0 kbDNA fragment downstream of the laeA stop cordon (primers FUM3 and FUM4)on either side of the A. parasiticus pyrG marker gene obtained frompBZ³⁷. pJW58 was used to disrupt the A. fumigatus laeA gene in strainAF293.1 to create TJW54.2. TABLE 2 Primers Restriction PrimerSequence^(a) sites LAB 1 ATCTACCTTTCTGGGCTCCTGG LAE2CGTGAAGAACTTGGCGFITGTAG LA2 GACGAGCTCGTGGAACAGTGGAAGGAAC SacI LA3GCGAAGCTTATGAACCGCATCAACCGA HindIII OEF GCTGTGAAGCTTTGTACCCTGTTTCGCCHindIII OER GATTTGAAGCTTTGCTGGCATGGAACGG HindIII MT1ATGCTGAAGCTtGGAAACTGGGAAAGGGGTC HindIII GFP2TGACGAATTCTCTTAATGGTTTCCTAGCCTG EcoRI GFP31 TGCGGAATTCATGAGCAAGGGCGAGGAAEcoRI GFP4 GGATGCCTCGAGTTTGTACAGCTCGTCCATGC XhoI GFP5AAGCAGCTCGAGTAAGAGCAAAAGGCGACCAC XhoI GF1GTAGCGAAGCTTGCCACCATGAGCAAGGGCG HindIII GF2CGGCGAATTCCTTGTACAGCTCGTCCATGC EcoRI GF3 TTTGGAATTCGTTTCGCCGCTGATGTTTGAGEcoRI FUM1 GCGCACTTCTTTGTTTTGCCCT FUM2 CATCGGAATTCTTTCTTGAGCGGCC EcoRIFUM3 TACCAGGATCCAAAACCTCTCGCCA BamHI FUM4 CATGACGGTAACTAAGGATTTGG

[0149] Secondary Metabolite Analysis.

[0150] Published procedures were used to extract and analyze ST⁶,gliotoxin¹¹, lovastatin⁹ and monocolin J⁹. Further details are availablein references cited herein. ST was extracted from either GMM 50 ml shakecultures inoculated with 10⁷ spores/ml grown for 60 hours or solid mediacultures spread with 10⁶ spores/plate grown for 5 days. Dried STextracts were resuspended in 100 μl chloroform and 10 μl was separatedin chloroform:acetone (8:2) on TLC plates. ST (Sigma Chem Co) wasspotted as a standard. MONJ was extracted from 50 ml GMM shake culturesinoculated with 10⁷ spores/ml grown for 72 hours. MONJ from WT:lov+ andOE::laeA;lov+ strains was extracted from cultures grown in 50 ml liquidshaking GMM for 14h at 37° C. and then transferred to liquid shakingthreonine minimal media (TMM) for 24h. Dried MONJ extracts wereresuspended in 100 μl methanol and 10 μL was separated in methanol:0.1%phoshoric acid (9:1) on C-18 reversed phase TLC plates. MONJ standardwas extracted from A. nidulans strain WMH1739 (Table 1). All experimentswere triplicated. Gliotoxin production in A. fumigatus was analyzed bymodification of the TLC method of Belkacemi et al.³⁸. Gliotoxin wasextracted from 50 ml GMM shake cultures inoculated with 10⁷ spores/mlgrown for 3 days. Dried chloroform extracts were resuspended in 100 μlmethanol and 10 μl was separated in chloroform:methanol (9:1). Gliotoxin(Sigma Chem Co.) was spotted as a standard. All experiments weretriplicated. To assess PN production, Micrococcus luteus ATCC 9341 wasgrown on TBS (Bacto Trypton 17 g, Bacto Soyton 3 g, NaCl 5 g, K₂HPO₄ 2.5g and glucose 2.5 g in 1 liter) at 37° C. 180 rpm until O.D.=1.3.3 ml ofM. luteus culture was mixed with 40 ml TSA (Bacto Trypton 15 g, BactoSoyton 5 g, NaCl 5 g, and agar 10 g in 1 liter) and poured in 150 cmdiameter plates to solidify. Fifty ml cultures of WT, ΔlaeA and OE::laeAstrains (10⁷ spores/ml) were grown in liquid shaking GMM for 14h at 37°C. and then transferred to liquid shaking LMM amended with 30 mMcyclopentanone for 24h. For each strain, six ml were removed,lyophilized and resuspended in 1 ml distilled water. One hundred μlsamples, with or without 6 units β-lactamase, were placed in 10 cm wellsof the M. luteus plates. Plates were placed for 2h at 4° C. and thenincubated over night at 37° C. to evaluate PN inhibition zones. Allexperiments were duplicated. Lovastatin was extracted from A. terreuscultures grown in 50 ml liquid shaking GMM for 18h at 32° C. and thentransferred to liquid shaking LMM with 30 mM cyclopentanone for 36h at32° C. Extraction and identification on TLC were followed by thepreviously described method in MONJ examination. Lovastatin (Merk. Co.)was spotted as a control. All experiments were duplicated.

[0151] B. Cloning of the laeA Gene and Characterization of the EncodedPolypeptide.

[0152] The greatest number of known fungal secondary metabolites hasbeen ascribed to the Ascomycete genus Aspergillus. Studies ofAspergillus nidulans have demonstrated the power of using a model systemto elucidate the molecular genetics of fungal secondary metabolism,principally penicillin (PN, an antibiotic) and sterigrnatocystin (ST, acarcinogen biochemically related to the agricultural contaminantaflatoxin) biosynthesis (reviewed in Brakhage² and Hicks et al.³respectively). These studies have established several characteristics offungal secondary metabolism including clustering of biosynthetic andregulatory genes as well as a genetic connection linking secondarymetabolite biosynthesis with sporulation through a shared signaltransduction pathway. The inventors were interested in identifyingglobal regulators of secondary metabolism in fungi that can uncouple thesporulation process from secondary metabolism production. Suchregulatory elements are extremely desirable because they would possessbroad specificity for the activation and/or repression of secondarymetabolite genes while providing strains capable of otherwise normal ornear-normal development and growth.

[0153] Previously a mutagenesis screen led to isolation of 23 mutantsdisplaying loss of ST production but normal sporulation in A. nidulans⁵. Three of the mutants were unable to express aflR that encodes a STcluster Zn₂Cys₆ transcription factor regulating ST biosynthetic geneexpression⁶. The inventors were able to complement one of these threemutants, RYJ8, with an A. nidulans trpC genomic cosmid library.Sequencing of a 4.5 kb subclone (pJW15) of the complementing cosmidpCOSJW3 revealed a 3 kb ORF designated as laeA (for loss of aflRexpression). Sequencing of the mutant allele, laeA1, from RYJ8 showed ithas a base pair transversion (1455; C->G) and a one base pair deletion(1453 bp) of the gene. The deletion resulted in a premature stop codon.Examination of genomic and cDNA sequence revealed that laeA has oneintron and three putative AflR binding sites⁶, one in the promoter(−607) and two in the encoding region (607 and 1487, FIG. 1a). cDNAanalysis showed laeA possesses a 5′ untranslated region (642 bp) (FIG.1a). Analysis of available genomic databases indicated only filamentousfungi have obvious LaeA homologs including A. fumigatus (human pathogen,aspergillosis, TIGR http://www.tigr.org/tdb/e2k1/afu1/), Neurosporacrassa (model fungus, GenBank™), Magnaporthe grisea (plant pathogen,rice blast fungus,http://www-genome.wi.mit.edu/annotation/fungi/magnaporthe), Coccidioidesimmitis (human pathogen, coccidioidomycosis, GenBank™) and Fusariumsporotrichioides (plant pathogen, trichothecene mycotoxin producer,http://www.genome.ou.edu/fsporo.html) (FIG. 1b). Examination of the LaeAamino acid sequence (375 amino acids) revealed a conserveds-adenosylmethionine (SAM) binding site found exclusively in nuclearprotein methyltransferases (FIG. 1b)⁷. Although the amino acid sequenceof LaeA did not show the presence of a nuclear localization motif, greenfluorescent protein (sGFP) tagging to either the 5′ or the 3′ end of A.nidulans laeA showed LaeA to be localized in the nucleus (FIG. 5).Biochemical analysis showed that LaeA methylated a ca. 30 kDa nuclearprotein (FIG. 1c).

[0154] laeA null mutants (ΔlaeA) were created by replacing laeA withmethG and pyrG in A. nidulans TJH3.40 (a methG1 auxotroph) and A.fumigatus AF293.1 (a pyrG auxotroph), respectively. Southern and PCRanalyses were carried out to confirm single gene replacement events inseveral transformants including A. nidulans TJW35.5 and A. fumigatusTJW54.2. Prototroph RJW46.4 was obtained from TJW35.5 by a sexual crossas described in the materials and methods section. RJW46.4 and TJW54.2were used for this study. In both spp., ΔlaeA strains were visuallydetectable due to loss of mycelial pigment in the backside of colonies(FIG. 2a and FIG. 6). An examination of organic extracts of A. nidulansand A. fumigatus ΔlaeA strains showed that production of severalmetabolites were reduced, including ST in A. nidulans (FIG. 2b) and theimmunotoxin gliotoxin in A. fumigatus (FIG. 2c). A verification thatthese defects were caused by loss of laeA was obtained by transformingA. nidulans ΔlaeA with wild type laeA and observing remediation ofmetabolite production.

[0155] To confirm the initial observation that laeA is required for STgene regulation, the inventors assessed aflR and stcU (a gene encoding abiosynthetic enzyme required for ST production)³ expression in the ΔlaeAbackground. Neither gene was expressed (FIG. 3a). A transcriptionalprofile of the entire ST gene cluster, which covers ca. 60 kb andcontains ca. 26 genes⁸, suggests LaeA transcriptional control is STcluster specific as transcription of upstream and downstream genes ofthe ST cluster are unaffected (FIG. 7). Because many uncharacterizedmetabolites were reduced in the ΔlaeA strains (FIG. 2b), the inventorsbelieved it possible that LaeA could be a global regulator for secondarymetabolite gene expression. To address this hypothesis, the inventorsexamined PN gene expression in the ΔlaeA strain. FIG. 3a shows that ipnA(a gene encoding isopenicillin N synthetase, a biosynthetic enzymerequired for PN biosynthesis)² expression was greatly reduced in theΔlaeA strain. The inventors' results of ST and PN gene expressionsuggested a role for LaeA in secondary metabolite gene clusterregulation. To further address this potential role, the inventorsexamined the expression of the heterologous LOV gene cluster in the A.nidulans ΔlaeA background. The partial LOV cluster, derived from A.terreus, was originally transformed into A. nidulans to study aspects oflovastatin biosynthesis⁹. This strain was used to cross the LOV cluster(LOV+) into appropriate laeA mutant backgrounds. FIG. 3b shows theΔlaeA; LOV+ strain displayed greatly diminished levels of both lovE (aLOV specific Zn₂Cys₆ transcription factor) and lovC (a LOV biosyntheticgene) transcripts. Organic extracts of this strain also showeddiminished production of monocolin J, MONJ (FIG. 3b), the LOVintermediate produced by the partial LOV cluster⁹.

[0156] The inventors next constructed laeA over expression strains(OE::laeA) in both A. nidulans and A. terreus to examine secondarymetabolite gene expression and product formation. As shown in FIG. 3c,ipnA, lovE, lovC but not stcU expression were remarkably elevated in theA. nidulans OE::laeA background. Secondary metabolite production wascorrelated with transcript levels. MONJ production was increased ˜400%and high levels of PN where produced during times when wild type showedno PN activity (FIG. 3d). ST levels remained the same as wild type inthe OE::laeA background (FIG. 3c). Over expression of the A. nidulanslaeA gene in the lovastatin producing fungus, A. terreus, led to400-700% increases in LOV (FIG. 3e).

[0157] The steady state levels of ST transcripts and product formationin the OE::laeA background in contrast to increased PN and LOVtranscripts and product formation suggested a unique interaction betweenlaeA and ST gene regulation. Due to the presence of three AflR bindingsites in the A. nidulans gene (FIG. 1a), the inventors thought itpossible that AflR might regulate laeA expression. As shown in FIG. 3f,over expression of aflR (OE::aflR) down regulates laeA expressionalthough elimination of aflR (ΔaflR) does not affect laeA transcript(FIG. 8). This indicates both negative (laeA) and positive (stc genes)⁶regulatory effects of AflR on gene transcription. To the inventors'knowledge, this is the first description of a putative secondarymetabolite feedback mechanism. As overproduction of ST negativelyaffects fungal growth the inventors speculate this feedback loop mayhave evolved as a fitness trait. In contrast, neither the promoter orencoding region of A. fumigatus laeA contained AflR binding sites norwas an aflR ortholog found in the genome. Initial examinations of theAspergillus ΔlaeA strains showed them to be more susceptible toneutrophil kill than wild type. Presumably this is due to loss of toxinsecondary metabolites or melanins, known virulence factors in severalfungal systems^(11, 12).

[0158] ST biosynthesis is regulated in A. nidulans via a signaltransduction pathway and many of the genes involved in this signalingpathway are known¹. Therefore, the inventors looked at the possibleinteractions with laeA of five signaling genes encoding two members of aheterotrimeric G protein (fadA and sfaD)^(13, 14), a regulator ofG-protein signaling protein regulating FadA activity (flbA)¹⁵, a cAMPdependent kinase (pkaA)¹⁶ and a Ras protein (rasA)⁷. laeA expression wasexamined in wild type and strains carrying the following alleles: ΔflbA,fadA^(G42R), ΔfadA, ΔsfaD, ΔpkaA, OE::pkaA, and OE::rasA^(G17A) (seeTable 1). mRNA analysis of these mutants showed that OE::pkaA andOE::rasA^(G17A) completely inhibited laeA expression (FIG. 3f) whereaslaeA transcription was not affected in any of the other strains (FIG.8). Presence of laeA transcript in the ΔflbA and fadA^(G42R) mutants(FIG. 8 and FIG. 9) shows that laeA is not sufficient for aflRexpression as aflR is not expressed in these strains¹⁸. FIG. 4 thuslydepicts the inventors' current understanding of LaeA involvement insecondary metabolite regulation.

[0159] The requirement of a kinase for laeA function is reminiscent—to adegree—of a Streptomyces global regulatory system involving the proteinAfsR. AfsR is a transcription factor regulating secondary metabolism inS. coelicolor but morphogenesis in S. griseus (contrast to the similarrole LaeA has in the three Aspergillus spp. examined here).Phosphorylation of AfsR enhances its activity¹⁹. Like AfsR, LaeAregulation is at the transcriptional level but functionally, LaeAappears most similar to histone (HMTase) or arginine methyltransferasesthat play important roles in regulating gene expression ineukaryotes^(7, 20, 21). An interesting aspect of HMTase regulation hasbeen the recent discovery that histone methylation plays a role indefining boundaries of euchromatic and heterochromatic chromosomaldomains such as in the mating locus of yeast and the beta-globin locusin mice^(22, 23). These findings suggest that histone methylation may beimportant in the regulation of gene cluster boundaries and may support arelationship with LaeA and histones in the regulation of secondarymetabolite gene clusters. Also, studies of gene regulation in the A.nidulans PN gene cluster and the A. nidulans nitrate utilization genecluster have shown that chromatin remodeling and/or DNA conformationalchanges are required for expression of genes in these clusters^(24, 25).It is possible that ΔlaeA strain may be impaired in a type of clusterregulation that could be revealed through biochemical tests addressingDNA conformational changes. Regardless of mechanism, and none isexpressly adopted herein, the identification of LaeA presents asignificant advance in understanding the complex regulation of secondarymetabolite production and provides a platform for biotechnologicalmanipulations of their production. Manipulation of LaeA in filamentousfungi enables the increased production of pharmaceuticals and theelimination of fungal toxins by providing improved strains of engineeredorganisms and also contributes to the broader understanding of molecularmechanisms by which secondary metabolites are produced.

[0160] Those skilled in the art will recognize, or be able to ascertainusing no more then routine experimentation, numerous equivalents to thespecific polypeptides, nucleic acids, methods, assays and reagentsdescribed herein. Such equivalents are considered to be within the scopeof this invention and covered by the following claims. All publications,patents, and patent applications cited herein are hereby incorporated byreference in their entirety for all purposes.

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[0163] 3. Hicks, J. K., Shimizu, K. & Keller N. P., in The Mycota,Kempken, F. & Bennett, Eds. (Spring-Verlag, 2002), Vol. XI, chap 4.

[0164] 4. Hawksworth, D. L. The magnitude of fungal diversity: the 1.5million species estimate revisited. Mycol. Res. 105, 1422-1432 (2001).

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[0174] 14. Rosén, S., Yu, J.-H. & Adams, T. H. The Aspergillus nidulanssfaD gene encodes a G protein beta subunit that is required for normalgrowth and repression of sporulation. EMBO J. 18, 5592-5600 (1999).

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[0197] 37. Skory, C. D., Chang, P. K., Cary, J. & Linz, J. E. Isolationand characterization of a gene from Aspergillus parasiticus associatedwith the conversion of versicolorin A to sterigmatocystin in aflatoxinbiosynthesis. Appl. Environ. Microbiol. 58, 3527-3537 (1992).

[0198] 38. Belkacemi, L., Barton, R. C., Hopwood, V. & Evans, E. G. V.Determination of optimum growth conditions for gliotoxin production byAspergillus fumigatus and development of a novel method for gliotoxindetection. Medical Mycology 37, 227-233 (1999).

[0199] 39. Adams, T. H. & Timberlake, W. E. Developmental repression ofgrowth and gene expression in Aspergillus. Proc. Natl. Acad. Sci. USA87, 5405-5409 (1990).

[0200] 40. Fernandes, M., Keller, N. P. & Adams, T. H. Sequence-specificbinding by Aspergillus nidulans AflR, a C6 zinc cluster proteinregulating mycotoxin biosynthesis. Mol. Microbiol. 28, 1355-1365 (1998).

[0201] 41. Kennedy, J. et al. Modulation of polyketide synthase activityby accessory proteins during lovastatin biosynthesis. Science 284,1368-1372 (1999).

[0202] 42. J. Sambrook, E. F. Fritsch, T. Maniatis, Molecular Cloning: ALaboratory Manual (Cold Spring Harbor Lab. Press, Plainview, N.Y.)(1989).

[0203] 43. Tilburn, J. et al. The Aspergillus PacC zinc fingertranscription factor mediates regulation of both acid- andalkaline-expressed genes by ambient pH. EMBO J. 14, 779-790 (1995).

[0204] 44. Yu, J.-H., Wieser, J. & Adams, T. H. The Aspergillus FlbA RGSdomain protein antagonizes G protein signaling to block proliferationand allow development. EMBO J. 15, 5184-5190 (1996).

[0205] 45. Shimizu, K. & Keller, N. P. Genetic involvement of acAMP-dependent protein kinase in a G protein signaling pathwayregulating morphological and chemical transitions in Aspergillusnidulans. Genetics 157, 591-600 (2001).

[0206] 46. Lee, B. N. & Adams, T. H. Overexpression of flbA, an earlyregulator of Aspergillus asexual sporulation, leads to activation ofbrlA and premature initiation of development. Mol. Microbiol. 14,323-334 (1994).

[0207] 47. Rosén, S., Yu, J.-H. & Adams, T. H. The Aspergillus nidulanssfaD gene encodes a G protein beta subunit that is required for normalgrowth and repression of sporulation. EMBO J. 18, 5592-5600 (1999).

[0208] 48 Brown D. W. et al. Twenty-five coregulated transcripts definea sterigmatocystin gene cluster in Aspergillus nidulans. Proc. Natl.Acad. Sci. USA 93, 1418-1422 (1996).

1 3 1 3100 DNA Aspergillus nidulans 1 tttaaagcac cgcagtagcg caataccaagcggaccgtcg tcgagctgga ctcgctcgac 60 tgccaggacg cactcgagga gggtcctgagtgctgaatct gatacaccac ttgcagaggc 120 tagcagctga cagcgccttc gtcgtgcagagtagtctagt agtctacagg ggcccgatcc 180 tagtcatcct agccatctta gccatcctaaccttcctagc cttcctagcc atcctagtgc 240 ctccgccagg cctttctcac ccccgatctcaaaaataata ataataataa aagaagtcga 300 cctcgtctgc ttttgcaact gcctcccgccccgccccctt cgcgcccgtg ggcccaaccg 360 ggtcaagaga gcggctgcga caatcataaacttaaagaaa agacggcgct gcttgcttgc 420 ttgcctgctt gcctgcttgc ttgcttgtctaaacgcccgt cctgttgttg tcccgcttcg 480 ctctctgttg gtttatgggt gtcagtccctcgcgctcttc cagcaccgtg accgctactt 540 tttctcagcc tgtcaatcgt caatcttcaatttcccttgt gggaggctct gattcccgta 600 taattgaccc ctcgccctcc tgcatcaatattcggattcc tgcttctatt caacgggcag 660 aatttcttcc agctcgtcca cccctcacttcgatgaacct ctcctttttc ttttcaatcg 720 gctgtttaca ttgtgttttc tgggatctctggacgagtca actagatctt cagcttctgg 780 attgatctaa cgaacattct ttcggcttctggccccccgc gctgctctgc ctacccgccg 840 cccagcgcac cccacggctt tggtgacgatttgtatagtc cactgttata gtccactgaa 900 aacagttcct tccactgttc cactcggctgtcgatctttg taccctgttt cgccgctgat 960 gtttgagatg ggcccggtgg gaactcgtctccccgccatg acctctccag cgcacaacca 1020 ctacagctac cactctccca cctccagcgacagaggccgg tcaaggcaga actcggatgc 1080 catggacatc cagtccatca ctgaacgagagccggcgacc agatacgcgg ttgcgggcgg 1140 ccctgcgccc tggaatcgca acgggtctccgagcatgagc cctatgtata gcaagtacat 1200 ctctcttacc cctccgtttc tttctgcttttctaccaccc catccctctt tccagtctga 1260 gtccaggctt gttccgcttg aagtggctaatgtgatcctc gtcttctctc tttctgtgtt 1320 ttagcaattc cgagcgaaac cagtttcatgaagagaacgg acgcacctac catggctttc 1380 gcaggggaat gtattttctt ccgtgcgatgagcaagaaca ggatcgcctc gacatcttcc 1440 ataagctatt cacggtagcg cgggtatcggagagtctgat ctacgcgccc catccaacca 1500 acggccggtt tctggaccta ggatgtggaactggtatctg ggcgatcgag gtagcgaaca 1560 agtaccctga tgcgtttgtc gctggtgtggatttggctcc tattcagcct ccgaaccacc 1620 cgaagaactg cgagttctac gcgcccttcgacttcgaagc gccatgggcc atgggggagg 1680 attcctggga tctaatccat ctgcagatgggttgcggtag tgtcatgggc tggccaaact 1740 tgtatcgaag gatattcgca catctccgtcccggtgcctg gtttgagcag gttgagatcg 1800 atttcgagcc tcgatgtgat gatcggtcactagatggaac ggcattgcgg cattggtacg 1860 attgtcttaa acaggcgaca gcagagaccatgcggccaat cgcccatagc tcccgcgata 1920 caataaaaga cctgcaggac gctgggttcacggagatcga ccatcaaata gtgggactcc 1980 cgctcaaccc gtggcatcag gacgaacacgagcggaaggt ggcacgttgg tataacctgg 2040 ccgtctcaga gagcatcgaa aacctcagtctggctccctt cagtcgtgtc tatcgctggc 2100 ccctggagag aatccagcaa ctcgccgcagatgtgaagtc cgaagcattc aacaaagaga 2160 tccatgccta caatatactg cacatataccaggctaggaa accattaaga taagagcaaa 2220 aggcgaccac atccaggaac gcaaaacgaaaaggaggaaa actgctagcg caagtttatg 2280 tcacgctggc acacgcccag ccatcagaaatctcaacagc gaaagttatg aaccgcatca 2340 accgagtatg aacgacaatt cgtccatcacacacccttcg gttcctctcg caggcccagc 2400 atggcgccct atcaacctgc tttacgacgtcgtatatact ggcgaagtat cctctctatc 2460 tactctggcg ctctagatac cgtgaagatgcagacaaaat tggccgagct cccttctcat 2520 aatcctcgac gccgcggggg tagtcgttatacgtgaaact atcatgacgc tcttgtcgtt 2580 accgctgcgc acctgggagg tataatagagtcgaattgcc ggtatcgtat actcatcgcg 2640 ggaatggaga tgatagagag ctatatccccggatcaagaa gttggccatg acggagtacg 2700 agaggagcat ggaaacaagc gacgagtaggtgagttatgt ggtgtggatc taggccccat 2760 atatattccc gagtcatgct aggtcccacactccggtttc tgcgacatat gcatgcaaca 2820 aagaatttgc ataaggcaat tgaaagctagtcacaaatag gagataaatt tatattgagc 2880 agaagcgaaa aggtttgact tctcgatccttcatgtttac gttgcctaaa ctccaacccg 2940 tgattgatac tatatggatc ccatgtacccgttccatgcc agcaaacaag caaatcccaa 3000 acgcctaaat ggctaggacc ggtccgtaagtctatgttta cagcttaaag gtggtcaaga 3060 gatgatccca tctcttactt gcgcagataattaccgtatc 3100 2 1125 DNA Aspergillus nidulans 2 atgtttgaga tgggcccggtgggaactcgt ctccccgcca tgacctctcc agcgcacaac 60 cactacagct accactctcccacctccagc gacagaggcc ggtcaaggca gaactcggat 120 gccatggaca tccagtccatcactgaacga gagccggcga ccagatacgc ggttgcgggc 180 ggccctgcgc cctggaatcgcaacgggtct ccgagcatga gccctatgta tagcaacaat 240 tccgagcgaa accagtttcatgaagagaac ggacgcacct accatggctt tcgcagggga 300 atgtattttc ttccgtgcgatgagcaagaa caggatcgcc tcgacatctt ccataagcta 360 ttcacggtag cgcgggtatcggagagtctg atctacgcgc cccatccaac caacggccgg 420 tttctggacc taggatgtggaactggtatc tgggcgatcg aggtagcgaa caagtaccct 480 gatgcgtttg tcgctggtgtggatttggct cctattcagc ctccgaacca cccgaagaac 540 tgcgagttct acgcgcccttcgacttcgaa gcgccatggg ccatggggga ggattcctgg 600 gatctaatcc atctgcagatgggttgcggt agtgtcatgg gctggccaaa cttgtatcga 660 aggatattcg cacatctccgtcccggtgcc tggtttgagc aggttgagat cgatttcgag 720 cctcgatgtg atgatcggtcactagatgga acggcattgc ggcattggta cgattgtctt 780 aaacaggcga cagcagagaccatgcggcca atcgcccata gctcccgcga tacaataaaa 840 gacctgcagg acgctgggttcacggagatc gaccatcaaa tagtgggact cccgctcaac 900 ccgtggcatc aggacgaacacgagcggaag gtggcacgtt ggtataacct ggccgtctca 960 gagagcatcg aaaacctcagtctggctccc ttcagtcgtg tctatcgctg gcccctggag 1020 agaatccagc aactcgccgcagatgtgaag tccgaagcat tcaacaaaga gatccatgcc 1080 tacaatatac tgcacatataccaggctagg aaaccattaa gataa 1125 3 374 PRT Aspergillus nidulans 3 MetPhe Glu Met Gly Pro Val Gly Thr Arg Leu Pro Ala Met Thr Ser 1 5 10 15Pro Ala His Asn His Tyr Ser Tyr His Ser Pro Thr Ser Ser Asp Arg 20 25 30Gly Arg Ser Arg Gln Asn Ser Asp Ala Met Asp Ile Gln Ser Ile Thr 35 40 45Glu Arg Glu Pro Ala Thr Arg Tyr Ala Val Ala Gly Gly Pro Ala Pro 50 55 60Trp Asn Arg Asn Gly Ser Pro Ser Met Ser Pro Met Tyr Ser Asn Asn 65 70 7580 Ser Glu Arg Asn Gln Phe His Glu Glu Asn Gly Arg Thr Tyr His Gly 85 9095 Phe Gly Gly Arg Met Tyr Phe Leu Pro Cys Asp Glu Gln Glu Gln Asp 100105 110 Arg Leu Asp Ile Phe His Lys Leu Phe Thr Val Ala Arg Val Ser Glu115 120 125 Ser Leu Ile Tyr Ala Pro His Pro Thr Asn Gly Arg Phe Leu AspLeu 130 135 140 Gly Cys Gly Thr Gly Ile Trp Ala Ile Glu Val Ala Asn LysTyr Pro 145 150 155 160 Asp Ala Phe Val Ala Gly Val Asp Leu Ala Pro IleGln Pro Pro Asn 165 170 175 His Pro Lys Asn Cys Glu Phe Tyr Ala Pro PheAsp Phe Glu Ala Pro 180 185 190 Trp Ala Met Gly Glu Asp Ser Trp Asp LeuIle His Leu Gln Met Gly 195 200 205 Cys Gly Ser Val Met Gly Trp Pro AsnLeu Tyr Arg Arg Ile Phe Ala 210 215 220 His Leu Arg Pro Gly Ala Trp PheGlu Gln Val Glu Ile Asp Phe Glu 225 230 235 240 Pro Arg Cys Asp Asp ArgSer Leu Asp Gly Thr Ala Leu Arg His Trp 245 250 255 Tyr Asp Cys Leu LysGln Ala Thr Ala Glu Thr Met Arg Pro Ile Ala 260 265 270 His Ser Ser ArgAsp Thr Ile Lys Asp Leu Gln Asp Ala Gly Phe Thr 275 280 285 Glu Ile AspHis Gln Ile Val Gly Leu Pro Leu Asn Pro Trp His Gln 290 295 300 Asp GluHis Glu Arg Lys Val Ala Arg Trp Tyr Asn Leu Ala Val Ser 305 310 315 320Glu Ser Ile Glu Asn Leu Ser Leu Ala Pro Phe Ser Arg Val Tyr Arg 325 330335 Trp Pro Leu Glu Arg Ile Gln Gln Leu Ala Ala Asp Val Lys Ser Glu 340345 350 Ala Phe Asn Lys Glu Ile His Ala Tyr Asn Ile Leu His Ile Tyr Gln355 360 365 Ala Arg Lys Pro Leu Arg 370

What is claimed is:
 1. An isolated polypeptide comprising an amino acidsequence selected from the group consisting of: (a) an amino acidsequence which is at least 85% identical to the amino acid sequence setforth in SEQ ID NO:3; (b) an amino acid sequence encoded by a nucleicacid comprising a nucleotide sequence set forth in SEQ ID NO:2; and (c)an amino acid sequence encoded by a nucleic acid which specificallyhybridizes under stringent conditions to either strand of a denatured,double-stranded nucleic acid comprising a nucleotide sequence set forthin SEQ ID NO:2.
 2. An isolated polypeptide according to claim 1 whereinsaid isolated polypeptide has secondary metabolite gene clusterregulating activity.
 3. An isolated polypeptide according to claim 1wherein said polypeptide regulates the activity of a lovastatin orpenicillin biosynthesis gene cluster.
 4. An isolated polypeptideaccording to claim 1 wherein said isolated polypeptide has proteinmethyltransferase activity.
 5. An isolated polypeptide according toclaim 1 comprising an amino acid sequence at least 95% identical to theamino acid sequence set forth in SEQ ID NO:3.
 6. An isolated polypeptideaccording to claim 1 comprising an amino acid sequence set forth in SEQID NO:3.
 7. An isolated nucleic acid comprising a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequence setforth in SEQ ID NO:2; (b) a nucleotide sequence encoding an amino acidsequence set forth in SEQ ID NO:3; and (c) a nucleotide sequence whichspecifically hybridizes under stringent conditions to either strand of adenatured, double-stranded nucleic acid having a nucleotide sequence setforth in SEQ ID NO:2.
 8. An isolated nucleic acid according to claim 7wherein said isolated nucleic acid encodes a polypeptide havingsecondary metabolite gene cluster regulating activity.
 9. An isolatednucleic acid according to claim 7 wherein said isolated nucleic acidregulates the activity of a lovastatin or penicillin biosynthesis genecluster.
 10. An isolated nucleic acid according to claim 6 wherein saidisolated nucleic acid encodes a polypeptide having proteinmethyltransferase activity.
 11. An expression vector comprising anisolated nucleic acid according to claim 7 wherein said isolated nucleicacid is in operative association with one or more regulatory elements.12. A transformed host cell or organism comprising an isolated nucleicacid according to claim
 7. 13. A transformed host cell or organismaccording to claim 12 wherein said transformed host cell is capable ofat least a two fold increase in production of a secondary metaboliterelative to non-transformed cells or organisms.
 14. A method ofpreparing an isolated polypeptide comprising LaeA or fragments thereof,comprising the step of culturing a transformed host cell or organism ofclaim 12 under conditions conducive to expression of the polypeptide,and recovering the expressed polypeptide from the cell or organism inisolated form.
 15. A method of detecting a nucleic acid encoding anamino acid sequence set forth in SEQ ID NO:3 in a biological samplecomprising the steps of: (a) hybridizing a complement of a nucleotidesequence which encodes an amino acid sequence as set forth in SEQ IDNO:3 to a nucleic acid material of a biological sample thereby forming ahybridization complex; and (b) detecting the hybridization complexwherein the presence of the complex correlates with the presence of anucleic acid encoding an amino acid sequence set forth in SEQ ID NO:3.16. A method of increasing the amount of a secondary metabolite producedin a cell or organism, comprising the steps of: (a) obtaining a cell oran organism capable of biosynthesizing a secondary metabolite; (b)transforming said cell or organism with a nucleic acid according toclaim 8; and (c) culturing said transformed cell or organism so that anincrease in production of the secondary metabolite occurs in thetransformed cell or organism as compared to a non-transformed cell ororganism.
 17. A method according to claim 16 wherein said cell ororganism is an Aspergillus species.
 18. A method according to claim 17wherein the Aspergillus species is A. nidulans or A. terreus.
 19. Amethod according to claim 17 wherein the secondary metabolite islovastatin or penicillin.
 20. A method according to claim 16 whereinsaid nucleic acid according to claim 8 overexpresses a polypeptidehaving secondary metabolite gene cluster regulating activity.
 21. Amethod of decreasing the production of a secondary metabolite in atransformed cell or organism, comprising the steps of: (a) obtaining atransformed cell or organism capable of biosynthesizing a secondarymetabolite, said transformed cell or organism having a defective laeAgene wherein the defective laeA gene is no longer biologically activeand expression of secondary metabolite gene clusters is reduced; and (b)culturing said transformed cell or organism so that a decrease inproduction of the secondary metabolite occurs in the transformed cell ororganism as compared to a non-transformed cell or organism.
 22. A methodaccording to claim 21 wherein the transformed cell or organism is A.parasiticus or A. flavus.
 23. A method of producing an isolatedsecondary metabolite, comprising steps of: (a) obtaining a cell or anorganism capable of biosynthesizing a secondary metabolite; (b)transforming said cell or organism with a nucleic acid according toclaim 8; (c) culturing said transformed cell or organism underconditions conducive to increasing production of the secondarymetabolite in the transformed cell or organism as compared to anon-transformed cell or organism; and (d) recovering said secondarymetabolite from the transformed cell or organism in an isolated form.24. A method according to claim 23 wherein said cell or organism is anAspergillus species.
 25. A method according to claim 24 wherein theAspergillus species is A. nidulans or A. terreus.
 26. A method accordingto claim 24 wherein the secondary metabolite is lovastatin orpenicillin.
 27. A method according to claim 23 wherein said nucleic acidaccording to claim 8 overexpresses a polypeptide having secondarymetabolite gene cluster regulating activity.
 28. A method foridentifying a novel secondary metabolite biosynthesis gene cluster in afungus, said method comprising steps of: (a) obtaining a transformedfungus having a disrupted laeA gene; (b) isolating a sample of nucleicacids from the transformed fungus of step (a), said sample of nucleicacids representative of the expressed genes of the transformed fungus;(c) hybridizing the sample of nucleic acids isolated in step (b) ornucleic acid equivalents of same with an array comprising a plurality ofnucleic acids representative of the expressed genes of a non-transformedfungus under conditions to form one or more hybridization complexes; (d)detecting said hybridization complexes; (e) comparing the levels of thehybridization complexes detected in step (c) with the level ofhybridization complexes detected in a sample of nucleic acids isolatedfrom an laeA-expressing fungus and representative of the expressed genesof the laeA-expressing fungus, wherein an altered level of hybridizationcomplexes detected in step (c) compared with a level of hybridizationcomplexes of the sample of nucleic acids from the laeA-expressing funguscorrelates with and identifies at least one gene under regulatorycontrol of the laeA gene product; and (f) examining genomic nucleotidesequence surrounding said gene identified in step (e) to determine ifsaid gene is clustered with secondary metabolite biosynthesis genesthereby identifying a novel secondary metabolite biosynthesis genecluster.
 29. A method according to claim 28, wherein said nucleic acidsrepresentative of the expressed genes of the non-transformed fungus instep (c) are immobilized on a substrate.
 30. A method according to claim28, wherein said said nucleic acids representative of the expressedgenes of the non-transformed fungus in step (c) are hybridizableelements in a microarray.
 31. A method according to claim 28, whereinthe sample of nucleic acids isolated from the laeA-expressing fungus andrepresentative of the expressed genes of the laeA-expressing fungus instep (e) are isolated from a transformed fungus overexpressing laeA geneproduct as compared to the non-transformed fungus.
 32. A methodaccording to claim 28 wherein the fungus is an Aspergillus or Fusariumspecies.